Transgenic mouse with a homozygous mutation in the CMAH gene

ABSTRACT

This application is in the field of sialic acid chemistry, metabolism, antigenicity, and the production of transgenic non-human mammals with altered sialic acid production. More particularly, this application relates to N-glycolylneuraminic acid (Neu5Gc) being an immunogen in humans, and the production of Neu5Gc-free mammalian products for laboratory and human use.

RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35U.S.C. 371, which claims priority to International Application No.PCT/US06/22282, filed Jun. 8, 2006, which application claims the benefitof priority to Provisional Application U.S. Ser. No. 60/688,867, whichwas filed on Jun. 8, 2005, the disclosures of which are incorporatedherein by reference.

GOVERNMENT INTERESTS

This invention was made with government support from the NationalInstitutes of Health under grant number R01-CA38701.

TECHNICAL FIELD

This application is in the field of sialic acid chemistry, metabolism,antigenicity, and the production of transgenic non-human mammals withaltered sialic acid production. More particularly, this applicationrelates to N-glycolylneuraminic acid (Neu5Gc) being an immunogen inhumans, and the production of Neu5Gc-free mammalian products forlaboratory and human use.

BACKGROUND OF THE INVENTION

All cells are covered with a dense and complex array of sugar chains.Sialic acids (Sias) are a family of nine-carbon sugars that aretypically present at the outermost units of these chains. By virtue oftheir terminal position, sialic acids act as binding sites for manyexogenous and endogenous receptors such as the Influenza viruses and theSiglic family of endogenous proteins. Such sugars are thus useful drugtargets for the prevention and treatment of infection. They are alsoinvolved in various biological and pathological processes such asneuronal plasticity and cancer metastasis. In many of these instances,the precise structures of the sialic acid and the residues it isattached to play critical roles. Thus, studying sialic acid functions isof great biological importance. In addition, sialic acids can be takenup from certain dietary sources (red meat and dairy products), and mayalso be associated with certain disease states, such as cancer and heartdisease.

Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)converts the sialic acid N-acetylneuraminic acid (Neu5Ac) toN-glycolylneuraminic acid (Neu5Gc.) In non-human mammals, Neu5Gc isrecognized by a number of endogenous binding proteins, as well as bypathogenic organisms such as bacteria and viruses. Humans are unable toproduce endogenous Neu5Gc because of an evolutionary inactivatingmutation in their CMAH gene. Specifically, this mutation involves aframe-shifting exon deletion of 92 base pairs in the 5′ region thatgives rise to a truncated protein that lacks the amino acid residuesthat are necessary for enzymatic activity (Schlenzka, W., et al., FEBSLett. (1996) 385: 197-200.) This mutation occurred sometime after thedivergence of humans from their last common ancestor, so humans are theonly known animals missing a functional CMAH gene (Chou, H-H, et al.,Proc. Nat. Acad. Sci. (2002), 99 (18): 11736-11741.) Although the causefor this mutation is unknown, it may have been caused by negativeselection of individuals that were CMAH+, because of the recognition ofNeu5Gc by pathogens.

Neu5Gc is known to be immunogenic in humans (Noguchi A., et al., J.Biochem. Tokyo (1995), 117 (1): 59-62.) Such immunogenicity is believedto play a role in the immune response observed in humans that come intocontact with mammalian products, such as cosmetics, food, mammaliancells and cell products, as well as therapeutic agents derived fromnon-human mammals or exposed to non-human mammalian products. Attemptshave been undertaken to try to diminish the Neu5Gc content ofrecombinantly produced human glycoproteins in cell lines by altering thecell lines using RNAi to suppress expression of the CMAH gene (Chenu S.,et al., Biochim. Biophys. Acta. (2003), 1622 (2): 133-144.)

However, there remains a need to produce biological products for humanuse that lack Neu5Gc, such as the production of human cells or tissuesin the absence of Neu5Gc medium, and by using transgenic non-humanmammals lacking a fully functional CMAH gene to produce Neu5Gc productsfor human use.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is provided a method forproducing animal products devoid of N-glycolneuraminic acid (Neu5Gc) forhuman use comprising the steps of: preparing a genetically alterednon-human mammal lacking a functional cytidine-5′-monophosphate N-acetylneuraminic acid hydrolase (CMAH) gene; and extracting at least oneanimal product from the genetically altered non-human animal (e.g.,ovine, bovine, piscine or porcine.)

The CMAH gene may be disrupted by a frame-shift mutation, or viaapplication of a cre-lox system mutation.

In specific examples, the CMAH gene may be mutated in the Rieskeiron-sulfur center to diminish gene function, such as at the site of theCys, His, or both Cys and His residues in the Rieske iron-sulfur center.Alternatively, the CMAH gene may be mutated in at least one of themononuclear iron center binding sites, in the CMP-Neu5Ac binding site,or in the cytochrome b5 binding site. All these sites are depicted inFIG. 16 and highly conserved in all animal species.

The non-human mammal product produced by the transgenic animal may beselected from the group consisting of: serum, muscle, tissue and milk.

The present invention also relates to a transgenic non-human mammalcomprising a CMAH gene lacking a functional copy of at least one of thegene domains associated with enzyme activity selected from the groupconsisting of: a Rieske Iron Sulfur center, a mononuclear iron centerbinding site, a CMP-Neu5Ac binding site, and a cytochrome b5 bindingsite; wherein said mammal produces animal products lacking Neu5Gc.

In one sense, the invention is simply a knockout non-human transgenicmammal that lacks expression of CMAH, which may be CMAH^(−/−).

The present invention also contemplates a non-human transgenic animalthat carries germline mutations in a CMAH gene that disrupt CMAHactivity, as well as a cell line derived from the knockout animal, whichmay be selected from the group consisting of: stem cells, epithelialcells and muscle cells.

Also included in the present invention is any animal product obtainedfrom the knockout non-human transgenic animal, which may be, forexample, tissue and serum.

In yet another embodiment, the present invention is a human embryonicstem cell line devoid of N-glycolylneuraminic acid (Neu5Gc) comprising:a preselected line of human embryonic stem cells; and a culture mediumcontaining Neu5Gc-non-human mammalian serum from a Neu5Gc nulltransgenic non-human mammal.

Other aspects of the invention are discussed throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the incorporation of free Neu5Gc in human epithelialcells. Caco-2 cells were fed or not fed for 3 days with Neu5Gc, ManNGcor Neu5Ac, each at 3 mM final concentration. The cells were harvestedand fractionated, and the Sia content of the different fractions ofcells was analyzed by 1,2-diamino-4,5-methylenedioxybenzene (DMB)derivatization and liquid chromatography analysis. FIG. 1(A) depicts theDMB-high pressure liquid chromatography (HPLC) profiles of Sia releasedfrom membrane fractions. Peaks indicated by an asterisk (*) correspondto Neu5Gc7Ac, Neu5,7Ac₂, Neu5,8,9Ac₃, Neu5Gc7,9Ac₂, and Neu5,7,9Ac₃,from left to right. Mass spectrometry (MS) and MS/MS data for the peakscorresponding to DMB-Neu5Gc and DMB-Neu5Ac from the membrane bound Siaof ManNGc and Neu5Gc-fed Caco-2 cells were also obtained (not shown.)FIG. 1(B) depicts the proportion of Neu5Gc (expressed as percent oftotal Sia) in the different fractions of Caco-2 cells. TH=totalhomogenate; HMW=high molecular weigh fraction; and LMW=cytosolic lowmolecular weight fraction.

FIG. 2 depicts free Neu5Gc taken up and incorporated into other types ofhuman cells. Human fibroblast and neuroblastoma cells were fed or notfed for 3 days with 3 mM Neu5Gc or Neu5Ac, and the cells were thenharvested and fractionated, and the Sia content in the differentfractions of cells was analyzed by DMB derivatization followed by HPLC.The proportion of Neu5Gc (expressed as percent of total Sia) of thedifferent fractions from (A) human fibroblasts or (B) humanneuroblastomas is shown. TH=total homogenate; HMW=high molecular weightfraction; and LMW=cytosolic low molecular weight fraction.

FIG. 3 depicts that the uptake of free Neu5Gc is not specific for humancells. Human and chimpanzee EBV-transformed lymphoblasts were fed or notfed for 3 days with 3 mM Neu5Gc or Neu5Ac, and the cells were thenharvested and fractionated, and the Sia content in the differentfractions of cells was analyzed by DMB derivatization followed by HPLC.The proportion of Neu5Gc (expressed as percent of total Sia) of thedifferent fractions from (A) human fibroblasts and (B) chimpanzeelymphoblasts is shown. TH=total homogenate; HMW=high molecular weightfraction; and LMW=cytosolic low molecular weight fraction.

FIG. 4 depicts that free Neu5Ac can compete with free Neu5Gc forincorporation into cells. Caco-2 cells grown in human serum were fed ornot fed for 3 days with 3 mM Neu5Gc with or without addition in themedia of Neu5Ac at 3 mM or 15 mM final concentration. The cells werethen harvested and fractionated. The Sia content in the differentfractions of cells was analyzed by DMB derivatization followed by HPLC.The proportion of Neu5Gc (expressed as percent of total Sia) of (A) thetotal homogenate fraction and (B) the membrane fraction is shown.

FIG. 5 depicts the uptake of free Neu5Gc by cells occurs via endocyticprocesses. Caco-2 cells grown in human serum were fed or not fed for 3days with 3 mM Neu5Gc in the presence or absence of various inhibitorsof endocytic pathways. The cells were then harvested and fractionated,and the Sia content in the different fractions of cells was analyzed byDMB derivatization followed by HPLC. The proportion of Neu5Gc (expressedas percent of total Sia) of (A) the total homogenate fraction and (B)the membrane fraction is shown.

FIG. 6 depicts that the lysosomal sialic acid transporter is involved inthe metabolic incorporation of free Neu5Gc. Wild-type (WT) and lysosomalsialic acid transporter mutant human fibroblasts (GM08496 and GM05520)grown in human serum were fed or not fed for 3 days with 3 mM Neu5Gc,ManNGc or Neu5Ac. The cells were then harvested and fractionated, andthe Sia content in the different fractions of cells was analyze by DMBderivatization followed by HPLC. The proportion of Neu5Gc (expressed aspercent of total Sia) in the membrane fraction is shown.

FIG. 7 depicts that both the lysosomal sialidase and the sialic acidtransporter are required for metabolic incorporation ofglycosidically-bound Neu5Gc from serum glycoconjugates. Wild type (WT)fibroblasts, lysosomal sialidase mutant fibroblasts (GM01718) and Siatransporter mutant fibroblasts (GM05520) were incubated with 10% fetalcalf serum (FCS) plus 20% horse serum (which are both rich sources ofglycosidically-bound Neu5Gc) for 3 days. Cells were then released fromthe flasks, fixed and analyzed for binding of a chicken anti-Neu5Gcantibody using a fluorescent labeled anti-chicken antibody, with andwithout permeabilization by flow cytometry. Neu5Gc-specific MFI is themedian fluorescence intensity (MFI) of the labeled anti-chicken antibodystaining with the background MFI subtracted out. At least 5000 cellswere counted for each staining. FIG. 7(A) depicts the Neu5Gc-specificMFI observed in all three different cell types. FIG. 7(B) depicts thepercent surface Neu5Gc that was calculated by dividing thenon-permeabilized Neu5Gc-specific MFI by the permeabilizedNeu5Gc-specific MFI.

FIG. 8 depicts proposed pathways for the uptake and incorporation ofNeu5Gc in human cells as published in Bardor, M., et al., J. Biol. Chem.(2005), 280 (6): 4228-4237.) The proposed model is based on the datapresented in this study and upon prior literature. The open diamondrepresents a Neu5Gc molecule; the shaded oval represents glypropteins;the open bullet represents the sialic acid transporter; and the doublezigzags represent ceramide. The thickness of the arrows also suggeststhe relative importance of various pathways in delivering Neu5Gc intothe cell. ST=sialyltransferase and TGN=trans-golgi network.

FIG. 9 depicts the detection of Neu5Gc on human embryonic stem cells(HESC) cultured under conventional conditions. Enhanced greenfluorescent protein (EGFP) transfected HESCs were grown on a murinefeeder layer in a medium containing 20% KNOCKOUT™ (Neu5Gc-rich) serumreplacement (Invitrogen Inc., Carlsbad, Calif., and described in WO98/30679.) a. HESCs were released with 2 mM EDTA and studies wereconducted by flow cytometry using a primary affinity-purified polyclonalchicken antibody specific for Neu5Gc, followed by a secondaryfluorescent labeled anti-chicken IgY antibody. The gray shaded plotrepresents the secondary antibody only; the thick line represents bothprimary and secondary antibody; and the thin line represents cellsincubated with the primary antibody in the presence of 1% chimpanzeeserum that contains Neu5Gc. b. HESCs were isolated byfluorescent-activated cell sorting (FACS) using the intrinsic EGFPfluorescence. Embryoid bodies (EBs) were derived by removing the feederlayer and growing the HESCs in reduced serum medium for 5 days. Bothtypes of cells (HESCs and EBs) were fractionated into membrane andcytosolic components. Sias were released and analyzed by DMBderivatization and HPLC. A peak corresponding to Neu5Gc is seen in allfractions.

FIG. 10 depicts that HESCs stably expressing EGFP can remainundifferentiated when Neu5Gc-deficient normal human serum (NHS) issubstituted for animal-derived culture medium components.

FIG. 11 depicts the effect of growth in normal human serum (NHS) oranimal-derived culture medium (Regular) on Neu5Gc content of HESCs andembryoid bodies (EBs).

FIG. 12 depicts the binding of “natural” antibodies from sera of normalhuman donors to HESCs. HESCs were grown in regular medium or in NHS for5 days. The cells were released with 2 mM EDTA and then exposed to serumfrom a human with a high level of anti-Neu5Gc antibodies (thick line) orfrom another individual with a low level of such antibodies (thin line)for 55 min., then stained with a secondary goat anti-human IgGconjugated to Alexa 594 dye (Molecular Probes, Eugene, Oreg.), andstudied by flow cytometry, with gating on the EGFP-positive HESCs. Thegray shaded plot shows the result with the secondary antibody alone.Immunoglobulin deposition was markedly reduced when cells were firstgrown in NHS-containing medium for 5 days (although non-specificbackground levels were increased.) The somewhat higher background seenwhen HESCs were grown in NHS-containing medium could be due to anon-specific IgG absorption, but it had no major consequences, such ascomplement deposition.

FIG. 13 depicts binding of complement C3b from human sera to EGFP+HESCs.HESCs were grown in regular medium or in NHS-containing medium for 5days and harvested with 2 mM EDTA. The cells were then exposed to anormal human serum from an individual with a high level of anti-Neu5Gcantibodies for 15 min. at 37° C., and also to the serum from anindividual with a low level of anti-Neu5Gc antibodies. Deposited C3b wasdetected using a goat anti-human C3b, then stained with a goatanti-human C3b conjugated to Alexa 594 and studied by flow cytometry. a.Control cells that where not exposed to any human sera showed verylittle (1%) positive staining for C3b. b. After exposure to the highlevel of anti-Neu5Gc antibodies serum, the double fluorescence plotshows that 37% of the EGFP+HESCs grown in regular medium showed positivestaining for human C3b. c. In contrast, only 13% of the EGFP+HESCsexposed to the low level of anti-Neu5Gc antibodies serum were positivefor C3b.

FIG. 14 depicts the targeting construct for inactivation of the CMAHenzyme in the mouse and final targeted Cmah allele. A 10 kb genomic DNAregion spanning the Cmah gene that includes the 92 base pairscorresponding to exon 6 of the murine Cmah was used to generate thetargeting construct pFlox-Ex-SL for elimination of CMAH enzyme activityby disabling this 92 base pair region that encodes the active site ofthe CMAH enzyme.

FIG. 15 depicts that CMAH null mice (Cmah−/−) are deficient in Neu5Gcexpression in their tissues, milk and plasma. Immunohistochemistry ofnumerous CMAH null tissues using a chicken anti-Neu5Gc and a secondaryhorseradish peroxidase (HRP) conjugated anti-chicken antibody showed noappreciable staining (data not shown.) Sialic acids were released by 2Macetic acid and derivatized with DM) and analyzed by DMB-HPL). As shown,Neu5Gc was only found in tissues from wild type mice. The absence ofNeu5Gc was confirmed using mass spectrometry (data not shown.)

FIG. 16 depicts a comparison of the deduced amino-acid sequence of theA. rubens CMP-Neu5Ac hydroxylase with several mammalian sequences. Thesequences were aligned with CLUSTLAW and subsequently shaded withGENEDOC. Residues identical in at least five of the six sequences areshaded grey, while homologous residues present in at least five of thesix sequences are printed white on dark grey. The hamster sequence isincomplete at the N- and C-termini. Box 1 indicates the binding site ofthe Rieske iron-sulfur center. Included in Box 1 are the critical Cys,His, Cys and His residues at positions 63 and 65 (at the beginning ofBox 1) and positions 84 and 87 at the end of Box 1. These are theliganding residues of the Rieske [2Fe2S]-cluster. Boxes 2 and 5 are thepostulated binding sites of a mononuclear iron center. Box 3 is thepostulated CMP-Neu5Ac binding site. Box 4 is the postulated site ofinteraction with cytochrome b5. Underlined amino acids indicate thepostulated transmembrane domain of the A. rubens hydroxylase. TheGenBank accession numbers of the sequences depicted are: mouse, D21826;hamster, AJ242835; pig, Y15010; chimpanzee, AF074481; and macque,AB013814. (Martensen, I., et al., Eur. J. Biochem. (2001) 268(19):5157-5166.)

FIG. 17 depicts the amino acid sequence of the human CMAH enzyme(UniProtKB/Swiss-Prot accession number Q9Y471. As shown, boxes 2 to 5that align with the sequences shown in FIG. 16 are identified. Also, asexpected, the approximately 92 amino acids that include the Box 1 Rieskeiron-sulfur center are missing. (Chou, H.-H., et al., Proc. Natl. Acad.Sci. USA (2002) 99:11736-11741.) Note that the same region is highlyconserved in all other species (see above.)

DETAILED DESCRIPTION OF THE INVENTION

This application is in the field of sialic acid chemistry, metabolism,antigenicity, and the production of transgenic non-human mammals withaltered sialic acid production. More particularly, this applicationrelates to N-glycolylneuraminic acid (Neu5Gc) being an immunogen inhumans, and the production of Neu5Gc-free mammalian products forlaboratory and human use.

The three related problems that are addressed by the present inventionare: 1) there is a need for Neu5Gc-free animal cell lines, which can beused to produce Neu5Gc biotherapeutic products; 2) there is a need forthe production of human cells and tissues for human use, as well as themaintenance of human organs for transplation, under Neu5Gc-freeconditions; and 3) there is a need for the production of transgenicNeu5Gc null mammals from which non-human animal products can be derivedfor human use.

Sialic Acid Chemistry and Metabolism

Sialic acid (Sia) is a generic name for a family of acidic nine carbonsugars typically found as the outermost units of glycan chains on thevertebrate cellular glycocalyx and on secreted glycoproteins. Theirlocation and widespread occurrence on all vertebrate cells allow them tobe involved in processes such as ligand-receptor interactions, cell-cellrecognition, cell-pathogen binding, inflammatory processes, immuneresponses and tumor metastases.

There are more than 50 kinds of Sias known in nature. Most are derivedvia biosynthetic modification of a Sia called N-acetylneuraminic acid(Neu5Ac). The addition of a single oxygen atom to the N-acetyl group ofNeu5Ac gives rise to a very common variation called N-glycolylneuraminicacid (Neu5Gc). The surfaces of most primate cell types studied to dateare dominated by these two major Sias.

Neu5Gc is perhaps the most widely expressed sialic acid in non-humanmammalian cells. While humans are genetically deficient in producingNeu5Gc, small amounts are present in human cells. A dietary origin wassuggested by human volunteer studies, and by observing that free Neu5Gcis metabolically incorporated into cultured human cells by unknownmechanisms. Research has shown that the incorporation of Neu5Gc maypredominantly originate from dietary sources (Tangvoranuntakul, P. etal. Proc. Natl. Acad. Sci. (USA) (2003) 100:12045-12050.) Red meat fromsources such as beef, pork and lamb are particularly rich in Neu5Gc andare likely the primary sources of Neu5Gc in the human diet. Also, dairyproducts contain Neu5Gc, although at somewhat lower levels than in redmeat.

In order for a Sia molecule to get attached to glycoconjugates, it mustfirst be activated by conversion to the sugar nucleotide derivative,cytidine-monophosphate-Sia (CMP-Sia). Thus, Sias are converted toCMP-Sias in the nucleus, which then return to the cytosol in order to betransported into the Golgi apparatus, where they serve as high-energydonors for attaching Sias to newly synthesized glycoconjugates on theirway to the cell surface. The biosynthetic transformation of Neu5Ac toNeu5Gc occurs at this sugar nucleotide level, wherein the CMP-Neu5Achydroxylase (CMAH) catalyzes the transfer of an oxygen atom toCMP-Neu5Ac, generating CMP-Neu5Gc. CMP-Neu5Gc can then be transportedinto the Golgi apparatus and used, in the same manner as CMP-Neu5Ac, toadd Neu5Gc to newly synthesized glycoconjugates. Indeed, these twonucleotide sugars appear to be used interchangeably by the Golgi CMP-Siatransporter and by the mammalian sialyltransferases, which transfer Siaresidues to cell surface and secreted glycoconjugates. Neu5Ac or Neu5Gcmolecules that are released from glycoconjugates during lysosomaldegradation processes can also be exported back into the cytosoliccompartment by a specific transporter. There, they are both available assubstrates for conversion to their respective CMP-Sia forms. Again,there appears to be no major difference in their conversion by CMP-Siasynthases. In this manner, Neu5Gc can be “recycled” for repeated use inGolgi sialation reactions.

It has been demonstrated that free Neu5Gc uptake occurs in a variety ofmammalian cells and tissues, such as secretory cells, cancer cells, andblood vessels. Inhibitors of certain non-clathrin mediated (i.e.receptor independent) endocytic pathways reduce Neu5Gc accumulation.Studies with human mutant cells show that the lysosomal sialic acidtransporter is required for metabolic incorporation of free Neu5Gc.Incorporation of glycosidically-bound Neu5Gc from exogenousglycoconjugates (relevant to human gut epithelial exposure to dietaryNeu5Gc) requires the transporter, as well as the lysosomal sialidase,which presumably acts to release free Neu5Gc. Thus, exogenous Neu5Gcreaches lysosomes via pinocytic/endocytic pathways, and is exported infree form into the cytosol, becoming available for activation andtransfer to glycoconjugates. In contrast, N-glycolylmannosamine (ManNGc)apparently traverses the plasma membrane by passive diffusion andbecomes available for conversion to Neu5Gc in the cytosol. Thismechanism can also explain the metabolic incorporation of chemicallysynthesized unnatural sialic acids.

Sialic Acid Genetics and Immunogenicity

Most normal healthy humans have a certain amount of circulatinganti-Neu5Gc antibodies, likely because of the fact that most humansingest food sources derived from non-human mammals containing highlevels of Neu5Gc. Thus, xenogenic (i.e., non-human) culturemethodologies may compromise implantation/transplantation success, dueto uptake and expression of Neu5Gc on the surface of any tissuedeveloped from human cells exposed to Neu5Gc-containing products. Thisproblem might also affect recombinant soluble biotherapeutic products.

Although Neu5Gc is a major Sia in most mammalian cells, it was longthought to be absent from healthy human tissues (Traving, C., et al.(1998) Cell. Mol. Life. Sci. 54: 1330-1349.) Indeed, humans aregenetically unable to synthesize Neu5Gc, due to an exon deletion/frameshift mutation in the human CMAH gene (Varki, A. (2002) Yearb. Phys.Anthropol. 44:54-69; Chou, H. H., et al. (1998) Proc. Ntl. Acad. Sci.USA 95:11751-11756; and Irie, A., et al. (1998) J. Biol. Chem. 273:15866-15871). It has been estimated that this mutation occurred in thehominid lineage—2.5 to 3 million years ago (Chou, H. H. et al. (2002)Proc. Natl. Acad. Sci. USA 99:11736-11741.) One dramatic consequence ofthis human-specific genetic defect appears to have been the suddenunmasking of the CD33-related Siglecs during human evolution, since theancestral condition of these molecules was to recognize Neu5Gc(Sonnengurg, J. L., et al. (2004) Glycobiology 14:339-346.)

Despite the absence of any known alternative pathway for the synthesisof Neu5Gc in humans, various groups have used antibodies to study theexpression of Neu5Gc in human tumors, particularly in various carcinomas(Hirabayashy, Y. et al. (1987) Jpn. J. Cancer Res. 78:251-260; Miyoshi,I. et al. (1986) Mol. Immunol. 23:631-638; Marquina, G. et al. (1996)Cancer Res. 56:5165-5171; Carr, A. et al. (2000) Hybridoma 19:241-247;Devine, P. L., et al. (1991) Cancer Res. 51:5826-5836; Kawachi S. et al.(1988) Int. Arch. Allergy Appl. Immunol. 85:381-383; and Higashi, H. etal. (1998) Jpn. J. Cancer Res. 79:952:956.) Recent studies havereexplored these findings, confirming prior reports of Neu5Gc expressionin human cancers and extending the finding to normal human tissues,including detecting small amounts of Neu5Gc in epithelial andendothelial cells of normal humans. Definitive confirmation resultedfrom releasing and purifying sialic acids from such tissues utilizing afluorescent derivatized form of Neu5Gc by HPLC and mass spectrometryanalysis (Tangvoranuntakal, P., et al. (2003) Proc. Natl. Acad. Sci. USA100:12045-12050.) Moreover, it was shown that exogenously added freeNeu5Gc is incorporated into cultured human carcinoma cells in vitro. Inaddition, oral ingestion studies of Neu5Gc in human volunteers werecarried out, providing evidence that the Neu5Gc found in human tissuesoriginates from dietary sources; particularly from red meat and milkproducts.

Because of the immunogenicity of Neu5Gc in humans, the production ofanimal products that lack Neu5Gc is, in some ways, half the story. Suchanimal products if they are to be acceptable for human use must alsolack anti-Neu5Gc antibodies which would be carried over to human hostsreceiving such animal products. Accordingly, it is desirable to limitthe amount of anti-Neu5Gc antibodies in such products. In one instance,it is desirable to limit the amount of anti-Neu5Gc antibodies to within10% (i.e., a “low” level of antibodies) of the level found in controlsystems that were prepared in the absence of a source of Neu5Gc. Theseexperiments are described in greater detail elsewhere herein.

Neu5Gc Null Mammals

The mammals that are used in the practice of the invention are thoseanimals generally regarded as useful for the production of mammalianproducts for human use, such as cosmetics, food stuffs, milk, mammaliancells and cell products, and therapeutic substances. Such mammalsinclude, for example, ovine such as lamb, bovine such as beef cattle andmilk cows, piscine and porcine, as well as rodents, such as mice andrats.

In one embodiment, the invention is a method to produce Neu5Gc-freetransgenic mammals and products therefrom comprising mutating the CMAHgene such that it produces CMAH with less or no activity, and therebyreducing or eliminating Neu5Gc from the biological material of thenon-human mammals. As detailed in FIGS. 16 and 17, the sequencesassociated with activity (depicted in the boxes) are well characterizedand highly conserved. Accordingly, the CMAH gene can easily be mutated,for example, by frame-shift mutation or the cre-lox system for deletionmutation, in addition to “knock-in” methods which would eliminateactivity, particularly if located in box 1. The biological materialderived from such mammals can be virtually any non-human organicmaterial which would otherwise contain Neu5Gc, such as a food stuffs(for example, red meat or a dairy product) or a mammalian derivedclinical sample used in human therapy, such as implanted cells orrecombinantly produced therapeutic proteins. The clinical sample may befrom any non-human mammalian source, such as ovine, bovine, piscine andporcine. Non-human clinical samples can be from any body fluid ortissue, such as serum, muscle tissue and milk, etc. The Neu5Gc-freemammals may be used to produce products for any use by humans, such ascultured human cells produced in laboratories, and cosmetics.

The same methodology used to knock out CMAH in mice is easily adaptedfor disruption of CMAH gene expression in domesticated animals, becauseof the high level of homology between CMAH genes in all mammals. Formice, a frameshift mutation, similar to the one found in the human CMAH,was introduced using the cre-lox recombination system. While normalwild-type mice express equal levels of Neu5Gc and Neu5Ac in their muscletissue, and approximately 5% Neu5Gc in their milk, the transgenic miceexhibit no evidence of Neu5Gc expression in tissues or milk Since themice are otherwise viable and fertile (as are humans), we can predictthat other CMAH null animals will also be the same.

The use of Neu5Gc-free products is useful in several commercialsettings. First, since consumption of Neu5Gc may pose a significant riskto human health, meat from Neu5Gc-free animals provides a saferalternative source of red meat. Second, as described in greater detailbelow, Neu5Gc-free serum can replace normal animal serum, which iscurrently used to culture human cells in laboratories. Third, the use ofNeu5Gc-free bovine products in cosmetics reduces the risk of immuneresponses against such products.

Culturing Human Cells in Neu5Gc-Free Medium

Considering that most humans also have antibodies to Neu5Gc,incorporation of Neu5Gc is hypothesized to be one of the factorscontributing to the health risks associated with high consumption of redmeat (such as heart disease and certain types of cancers). In addition,the presence of Neu5Gc in human cells that are cultured in animalproducts for use in human therapeutic agents is also a potential sourceof allergenicity. More particularly, when human cells are cultured inserum from animals, they can take up and incorporate Neu5Gc, potentiallyresulting in immunological rejection if such cells are used for therapy(e.g., transplantation of human embryonic stem cell-derived grafts.) Ithas now been demonstrated that targeted disruption of the CMAH gene inmice completely abolished the expression of Neu5Gc in all tissues aswell as in their secretions. Similar disruption of the CMAH gene indomesticated livestock (cows, pigs, goats, etc.) may provide a source ofNeu5Gc-free animal products, which are commonly used as researchmaterials (e.g., serum and cell extracts), as well as in cosmetics.

As described above, by targeting one single gene, CMAH, Neu5Gc can beeliminated from all mammalian tissues and secretions, including serum,muscle tissue and milk. Since Neu5Gc is immunogenic to humans,eliminating CMAH from domesticated animals would provide a source ofnon-immunogenic Neu5Gc-free products for human cell culture, tissueculture, and even organ preservation. For example, a CMAH null cow willnot uptake and incorporate Neu5Gc from ingested meat, milk etc.Accordingly, this invention provides a way of preparing transgenicanimals whose serum and other products can be used to produce cellcultures and tissues that will reduce the risk of developing potentiallyautoreactive antibodies after implantation of such cells and tissuesinto humans. The absence of Neu5Gc in mammalian serum products used forhuman cell tissue culture would also provide more human-like growthconditions.

HESCs can potentially generate every body cell type, making themexcellent candidates for cell and tissue replacement therapies. HESCsare typically cultured with animal-derived “serum replacements” onmurine feeder layers. Both of these are sources of the non-human sialicacid Neu5Gc, against which many humans have circulating antibodies. BothHESC and derived embryoid bodies metabolically incorporate significantamounts of Neu5Gc under standard conditions. Exposure to human sera withanti-Neu5Gc antibodies results in binding of immunoglobulin anddeposition of complement, which leads to cell killing in vivo. Levels ofNeu5Gc on HESCs and embryoid bodies dropped after culture inheat-inactivated anti-Neu5Gc-antibody-negative human serum, reducingbinding of antibodies and complement from high titer sera, whileallowing maintenance of the undifferentiated state. Absent theavailability of Neu5Gc-free mammalian products, complete elimination ofNeu5Gc would likely require using human serum with human feeder layers,ideally starting with fresh HESCs that have never been exposed to animalproducts.

The pluripotent abilities of HESCs have potential for treating manydiseases by transplantation of HESC-derived tissues. While safety is amajor issue regarding infection or tumorigenicity, the possibility ofrejection is also of concern. Current culture methods using animalproducts also carry the risk of infection by non-human pathogens. HESClines are traditionally cultured on mitotically-inactivated mouseembryonic fibroblasts (so-called “feeder layers”), and in a mediacontaining fetal calf serum. To avoid animal serum, certain proprietaryserum replacements are sometimes used. However, these also containanimal products. When HESCs are removed from the feeder layer and grownin suspension, they differentiate into aggregates called embryoid bodies(EB). EBs are formed by precursors of several cell lineages and can beinduced to differentiate into many cell types. Although the feeder layeris no longer necessary, EBs must still be maintained in “serumreplacement” medium, which likewise may contain Neu5Gc positive animalproducts.

Production of Transgenic Non-Human Animals.

The production of transgenic non-human animals is now a common methodused in the laboratory to alter the metabolism of various animals foruse as models of particular disease states. For instance, there areinsulin free mice, immunologically deficient animals of many species andthe like. Accordingly, such methods are commonly practice by those ofskill in the art and could easily be adapted to the teachings herein toproduce any animal models without undue experimentation. This isespecially true since the critical sequences of the HESC gene associatedwith its active site are well characterized and highly conserved.

In one embodiment, the present invention provides knockout non-humanmammals lacking a functional CMAH. “Knock-out” refers to partial orcomplete suppression of the expression of a protein encoded by anendogenous DNA sequence in a cell. The “knock-out” can be affected bytargeted deletion of the whole or part of a gene encoding a protein inan embryonic stem cell. As a result, the deletion may prevent or reducethe expression of the protein in any cell in the whole animal in whichit is normally expressed. For example, a “CMAH knock-out animal” refersto an animal in which the expression CMAH has been reduced or suppressedby the introduction of a recombinant nucleic acid molecule that lacks atleast a portion of the genomic DNA sequence encoding CMAH.

“Transgenic animal” refers to an animal to which exogenous DNA has beenintroduced while the animal is still in its embryonic stage. In mostcases, the transgenic approach aims at specific modifications of thegenome, e.g., by introducing whole transcriptional units into thegenome, or by up- or down-regulating pre-existing cellular genes. Thetargeted character of certain of these procedures sets transgenictechnologies apart from experimental methods in which random mutationsare conferred to the germline, such as administration of chemicalmutagens or treatment with ionizing solution.

The term “knockout mammal” and the like, refers to a transgenic mammalwherein a given gene has been suppressed by recombination with atargeting vector. It is to be emphasized that the term is intended toinclude all progeny generations. Thus, the founder animal and all F1,F2, F3, and so on, progeny thereof are included.

The term “chimera,” “mosaic,” “chimeric mammal” and the like, refers toa transgenic mammal with a knockout in some of its genome-containingcells.

The term “heterozygote,” “heterozygotic mammal” and the like, refers toa transgenic mammal with a disruption on one of a chromosome pair in allof its genome containing cells.

The term “homozygote,” “homozygotic mammal” and the like, refers to atransgenic mammal with a disruption on both members of a chromosome pairin all of its genome-containing cells.

A “non-human mammal” of the invention includes mammals such as rodents,primates, sheep, dogs (ovine such as lamb, bovine such as beef cattleand milk cows, piscine and porcine.)

Although the invention uses a typical non-human rodent animal (e.g.,rats and mice), other mammals can similarly be genetically modifiedusing the methods and compositions of the invention.

A “mutation” is a detectable change in the genetic material in theanimal, which is transmitted to the animal's progeny. A mutation isusually a change in one or more deoxyribonucleotides, the modificationbeing obtained by, for example, adding, deleting, inverting, orsubstituting nucleotides.

Typically, the genome of the transgenic non-human animal comprises oneor more deletions in one or more exons of the genes as depicted in theboxes in FIG. 16.

In principle, knockout animals may have one or both copies of the genesequence of interest disrupted. In the latter case, in which ahomozygous disruption is present, the mutation is termed a “null”mutation. In the case where only one copy of the nucleic acid sequenceof interest is disrupted, the knockout animal is termed a “heterozygousknockout animal”. The knockout animals of the invention are typicallyhomozygous for the disruption of both CMAH genes being targeted.

It is important to note that it is not necessary to disrupt a gene togenerate a transgenic organism lacking functional expression. Theinvention includes the use of antisense molecules that are transformedinto a cell, such that production of an OAT polypeptide is inhibited.Such an antisense molecule is incorporated into a germ cell as describedmore fully herein operably linked to a promoter such that the antisenseconstruct is expressed in all cells of a transgenic organism.

Techniques for obtaining the transgenic animals of the invention arewell known in the art. The techniques for introducing foreign DNAsequences into the mammalian germ line were originally developed inmice. One route of introducing foreign DNA into a germ line entails thedirect microinjection of linear DNA molecules into a pronucleus of afertilized one-cell egg. Microinjected eggs are subsequently transferredinto the oviducts of pseudopregnant foster mothers and allowed todevelop. About 25% of the progeny mice inherit one or more copies of themicro-injected DNA. Currently, the most frequently used techniques forgenerating chimeric and transgenic animals are based on geneticallyaltered embryonic stem cells or embryonic germ cells. Techniquessuitable for obtaining transgenic animals have been amply described. Asuitable technique for obtaining completely ES cell derived transgenicnon-human animals is described in WO 98/06834.

Knockout animals of the invention can be obtained by standard genetargeting methods as described above, typically by using ES cells. Thus,the invention relates to a method for producing a knockout non-humanmammal comprising (i) providing an embryonic stem (ES) cell from therelevant animal species comprising an intact CMAH gene; (ii) providing atargeting vector capable of disrupting the intact CMAH gene; (iii)introducing the targeting vector into the ES cells under conditionswhere the intact CMAH undergoes homologous recombination with thetargeting vector to produce a mutant CMAH gene; (iv) introducing the EScells carrying a disrupted CMAH gene into a blastocyst; (v) implantingthe blastocyst into the uterus of pseudopregnant female; (vi) deliveringanimals from said females, identifying a mutant animal that carries themutant allele and (vii) selecting for knockout animals and breedingthem.

A “targeting vector” is a vector comprising sequences that can beinserted into the gene to be disrupted, e.g., by homologousrecombination. The targeting vector generally has a 5′ flanking regionand a 3′ flanking region homologous to segments of the gene of interest,surrounding a foreign DNA sequence to be inserted into the gene. Forexample, the foreign DNA sequence may encode a selectable marker, suchas an antibiotics resistance gene. Examples for suitable selectablemarkers are the neomycin resistance gene (NEO) and the hygromycinP-phosphotransferase gene. The 5′ flanking region and the 3′ flankingregion are homologous to regions within the gene surrounding the portionof the gene to be replaced with the unrelated DNA sequence. DNAcomprising the targeting vector and the native gene of interest arecontacted under conditions that favor homologous recombination. Forexample, the targeting vector and native gene sequence of interest canbe used to transform embryonic stem (ES) cells, in which they cansubsequently undergo homologous recombination.

Thus, a targeting vector refers to a nucleic acid that can be used todecrease or suppress expression of a protein encoded by endogenous DNAsequences in a cell. In a simple example, the knockout construct iscomprised of a CMAH polynucleotide with a deletion in a critical portionof the polynucleotide (e.g., the 5′ terminus of the CMAH gene) so that afunctional CMAH cannot be expressed therefrom. Alternatively, a numberof termination codons can be added to the native polynucleotide to causeearly termination of the protein or an intron junction can beinactivated. In a typical knockout construct, some portion of thepolynucleotide is replaced with a selectable marker (such as the neogene) so that the polynucleotide can be represented as follows: CMAH5′/neo/CMAH 3′, where CMAH 5′ and CMAH 3′, refer to genomic or cDNAsequences which are, respectively, upstream and downstream relative to aportion of the CMAH polynucleotide and where neo refers to a neomycinresistance gene.

Proper homologous recombination can be confirmed by Southern blotanalysis of restriction endonuclease digested DNA using, as a probe, anon-disrupted region of the gene. Since the native gene, will exhibit arestriction pattern different from that of the disrupted gene, thepresence of a disrupted gene can be determined from the size of therestriction fragments that hybridize to the probe.

In an animal obtained by the methods above, the extent of thecontribution of the ES cells that contain the disrupted CMAH gene to thesomatic tissues of the transgenic animal can be determined visually bychoosing animal strains for the source of the ES cells and blastocystthat have different coat colors.

The transgenic animals can contain a transgene, such as reporter gene,under the control of a CMAH promoter or fragment thereof. Methods forobtaining transgenic and knockout non-human animals are known in theart. Knock out mice are generated by homologous integration of a“targeting vector” construct into a mouse embryonic stem cell chromosomewhich encodes a gene to be knocked out. In one embodiment, genetargeting, which is a method of using homologous recombination to modifyan animal's genome, can be used to introduce changes into culturedembryonic stem cells. By targeting a CMAH gene of interest in ES cells,these changes can be introduced into the germlines of animals togenerate chimeras. The gene targeting procedure is accomplished byintroducing into tissue culture cells a DNA targeting vector thatincludes a segment homologous to a target CMAH locus, and which alsoincludes an intended sequence modification to the CMAH genomic sequence(e.g., insertion, deletion, point mutation.) The treated cells are thenscreened for accurate targeting to identify and isolate those which havebeen properly targeted.

Generally, the embryonic stem cells (ES cells) used to produce theknockout animals will be of the same species as the knockout animal tobe generated. Thus for example, mouse embryonic stem cells will usuallybe used for generation of knockout mice.

Embryonic stem cells are generated and maintained using methods wellknown to the skilled artisan such as those described by Doetschman etal. (1985), J. Embryol. Exp. Mol. Biol. 87:27-45. Any line of ES cellscan be used, however, the line chosen is typically selected for theability of the cells to integrate into and become part of the germ lineof a developing embryo so as to create germ line transmission of theknockout construct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog no. CKL 1934). Still another ES cell line is the WW6 cell line(Ioffe et al. (1995) Proc. Nat. Acad. Sci. 92:7357-7361.) The cells arecultured and prepared for knockout construct insertion using methodswell known to the skilled artisan, such as those set forth in:Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. IRL Press, Washington, D.C. ([1987]; Bradley et al.(1986) Current Topics in Devel. Biol. 20:357-371; and Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1986).

A targeting vector construct refers to a uniquely configured fragment ofnucleic acid which is introduced into a stem cell line and allowed torecombine with the genome at the chromosomal locus of the gene ofinterest to be mutated. Thus, a given knock out construct is specificfor a given gene to be targeted for disruption. Nonetheless, many commonelements exist among these constructs and these elements are well knownin the art. A typical targeting vector contains nucleic acid fragmentsof not less than about 0.5 kb nor more than about 10.0 kb from both the5′ and the 3′ ends of the genomic locus which encodes the gene to bemutated. These two fragments are separated by an intervening fragment ofnucleic acid which encodes a positive selectable marker, such as theneomycin resistance gene (neo). The resulting nucleic acid fragment,consisting of a nucleic acid from the extreme 5′ end of the genomiclocus linked to a nucleic acid encoding a positive selectable markerwhich is in turn linked to a nucleic acid from the extreme 3′ end of thegenomic locus of interest, omits most of the coding sequence for CMAH orother gene of interest to be knocked out. When the resulting constructrecombines homologously with the chromosome at this locus, it results inthe loss of the omitted coding sequence, otherwise known as thestructural gene, from the genomic locus. A stem cell in which such ahomologous recombination event has taken place can be selected for byvirtue of the stable integration into the genome of the nucleic acid ofthe gene encoding the positive selectable marker and subsequentselection for cells expressing this marker gene in the presence of anappropriate drug (neomycin in this example).

Variations on this basic technique also exist and are well known in theart. For example, a “knock-in” construct refers to the same basicarrangement of a nucleic acid encoding a 5′ genomic locus fragmentlinked to nucleic acid encoding a positive selectable marker which inturn is linked to a nucleic acid encoding a 3′ genomic locus fragment,but which differs in that none of the coding sequence is omitted andthus the 5′ and the 3′ genomic fragments used were initially contiguousbefore being disrupted by the introduction of the nucleic acid encodingthe positive selectable marker gene. This “knock-in” type of constructis thus very useful for the construction of mutant transgenic animalswhen only a limited region of the genomic locus of the gene to bemutated, such as a single exon, is available for cloning and geneticmanipulation. Alternatively, the “knock-in” construct can be used tospecifically eliminate a single functional domain of the targeted gene,resulting in a transgenic animal which expresses a polypeptide of thetargeted gene which is defective in one function, while retaining theunction of other domains of the encoded polypeptide. This type of“knock-in” mutant frequently has the characteristic of a so-called“dominant negative” mutant because, especially in the case of proteinswhich homomultimerize, it can specifically block the action of (or“poison”) the polypeptide product of the wild-type gene from which itwas derived. In a variation of the knock-in technique, a marker gene isintegrated at the genomic locus of interest such that expression of themarker gene comes under the control of the transcriptional regulatoryelements of the targeted gene. One skilled in the art will be familiarwith useful markers and the means for detecting their presence in agiven cell.

As mentioned above, the homologous recombination of the above described“knock out” and “knock in” constructs is sometimes rare and such aconstruct can insert nonhomologously into a random region of the genomewhere it has no effect on the gene which has been targeted for deletion,and where it can potentially recombine so as to disrupt another genewhich was otherwise not intended to be altered. Such non-homologousrecombination events can be selected against by modifying theabove-mentioned targeting vectors so that they are flanked by negativeselectable markers at either end (particularly through the use of twoallelic variants of the thymidine kinase gene, the polypeptide productof which can be selected against in expressing cell lines in anappropriate tissue culture medium well known in the art, i.e. onecontaining a drug such as 5-bromodeoxyuridine.) Non-homologousrecombination between the resulting targeting vector comprising thenegative selectable marker and the genome will usually result in thestable integration of one or both of these negative selectable markergenes and hence cells which have undergone nonhomologous recombinationcan be selected against by growth in the appropriate selective media(e.g. media containing a drug such as 5-bromodeoxyuridine for example.)Simultaneous selection for the positive selectable marker and againstthe negative selectable marker will result in a vast enrichment forclones in which the knock out construct has recombined homologously atthe locus of the gene intended to be mutated. The presence of thepredicted chromosomal alteration at the targeted gene locus in theresulting knock out stem cell line can be confirmed by means of Southernblot analytical techniques which are well known to those familiar in theart. Alternatively, PCR can be used.

Each targeting vector to be inserted into the cell is linearized.Linearization is accomplished by digesting the DNA with a suitablerestriction endonuclease selected to cut only within the vector sequenceand not the 5′ or 3′ homologous regions or the selectable marker region.

For insertion, the targeting vector is added to the ES cells underappropriate conditions for the insertion method chosen, as is known tothe skilled artisan. For example, if the ES cells are to beelectroporated, the ES cells and targeting vector are exposed to anelectric pulse using an electroporation machine and following themanufacturer's guidelines for use. After electroporation, the ES cellsare typically allowed to recover under suitable incubation conditions.The cells are then screened for the presence of the targeting vector asexplained herein. Where more than one construct is to be introduced intothe ES cell, each targeting vector can be introduced simultaneously orone at a time.

After suitable ES cells containing the knockout construct in the properlocation have been identified by the selection techniques outlinedabove, the cells can be inserted into an embryo. Insertion may beaccomplished in a variety of ways known to the skilled artisan, howeverthe typical method is by microinjection. For microinjection, about 10-30cells are collected into a micropipet and injected into embryos that areat the proper stage of development to permit integration of the foreignES cell containing the recombination construct into the developingembryo. For instance, the transformed ES cells can be microinjected intoblastocytes. The suitable stage of development for the embryo used forinsertion of ES cells is very species dependent, however for mice it isabout 3.5 days. The embryos are obtained by perfusing the uterus ofpregnant females. Suitable methods for accomplishing this are known tothe skilled artisan.

While any embryo of the right stage of development is suitable for use,typical embryos are male. In mice, the typical embryos also have genescoding for a coat color that is different from the coat color encoded bythe ES cell genes. In this way, the offspring can be screened easily forthe presence of the knockout construct by looking for mosaic coat color(indicating that the ES cell was incorporated into the developingembryo.) Thus, for example, if the ES cell line carries the genes forwhite fur, the embryo selected will carry genes for black or brown fur.

After the ES cell has been introduced into the embryo, the embryo may beimplanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent. For mice, this stage is about2-3 days pseudopregnant.

Offspring that are born to the foster mother may be screened initiallyfor mosaic coat color where the coat color selection strategy (asdescribed above, and in the examples) has been employed. In addition, oras an alternative, DNA from tail tissue of the offspring may be screenedfor the presence of the knockout construct using Southern blots and/orPCR as described above. Offspring that appear to be mosaics may then becrossed to each other, if they are believed to carry the knockoutconstruct in their germ line, in order to generate homozygous knockoutanimals. Homozygotes may be identified by Southern blotting ofequivalent amounts of genomic DNA from mice that are the product of thiscross, as well as mice that are known heterozygotes and wild type mice.

Other means of identifying and characterizing the knockout offspring areavailable. For example, Northern blots can be used to probe the mRNA forthe presence or absence of transcripts encoding either the gene knockedout, the marker gene, or both. In addition, Western blots can be used toassess the level of expression of the CMAH gene knocked out in varioustissues of the offspring by probing the Western blot with an antibodyagainst the particular CMAH protein, or an antibody against the markergene product (i.e., the presence of Neu5GC using an antibody asidentified in PCT application no. PCT/US2004/022415.) Finally, in situanalysis (such as fixing the cells and labeling with antibody) and/orFACS (fluorescence activated cell sorting) analysis of various cellsfrom the offspring can be conducted using suitable antibodies to lookfor the presence or absence of the knockout construct gene product.

Yet other methods of making knock-out or disruption transgenic animalsare also generally known. See, for example, Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of an OAT gene can becontrolled by recombinase sequences.

Animals containing more than one knockout construct and/or more than onetransgene expression construct are prepared in any of several ways. Atypical manner of preparation is to generate a series of mammals, eachcontaining one of the desired transgenic phenotypes. Such animals arebred together through a series of crosses, backcrosses and selections,to ultimately generate a single animal containing all desired knockoutconstructs and/or expression constructs, where the animal is otherwisecongenic (genetically identical) to the wild type except for thepresence of the knockout construct(s) and/or transgene(s).

In another aspect, a transgenic animal can be obtained by introducinginto a single stage embryo a targeting vector. The zygote is the besttarget for micro-injection. In the mouse, the male pronucleus reachesthe size of approximately 20 micrometers in diameter which allowsreproducible injection of 1-2 pL of DNA solution. The use of zygotes asa target for gene transfer has an advantage in that in most cases theinjected DNA will be incorporated into the host gene before the firstcleavage (Brinster et al. (1985) Proc. Nat. Acad. Sci. 82:4438-4442.) Asa consequence, all cells of the transgenic animal will carry theincorporated nucleic acids of the targeting vector. This will in generalalso be reflected in the efficient transmission to offspring of thefounder since 50% of the germ cells will harbor the transgene.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear. At about this time, the nucleotide sequence comprisingthe trans gene is introduced into the female or male pronucleus. In somespecies such as mice, the male pronucleus is typically used. Typicallythe exogenous genetic material be added to the male DNA complement ofthe zygote prior to its being processed by the ovum nucleus or thezygote female pronucleus. It is thought that the ovum nucleus or femalepronucleus release molecules which may affect the male DNA complement,perhaps by replacing the protamines of the male DNA with histones,thereby facilitating the combination of the female and male DNAcomplements to form the diploid zygote.

Thus, the exogenous genetic material is typically added to the malecomplement of DNA or any other complement of DNA prior to its beingaffected by the female pronucleus. For example, the exogenous geneticmaterial is added to the early male pronucleus, as soon as possibleafter the formation of the male pronucleus, which is when the male andfemale pronuclei are well separated and both are located close to thecell membrane. Alternatively, the exogenous genetic material could beadded to the nucleus of the sperm after it has been induced to undergodecondensation. Sperm containing the exogenous genetic material can thenbe added to the ovum or the decondensed sperm could be added to the ovumwith the transgene constructs being added as soon as possiblethereafter.

Introduction of the a exogenous nucleic acid (e.g., a targeting vector)into the embryo may be accomplished by any means known in the art suchas, for example, microinjection, electroporation, or lipofection.Following introduction of the exogenous nucleic acid into the embryo,the embryo may be incubated in vitro for varying amounts of time, orreimplanted into the surrogate host, or both. In vitro incubation tomaturity is within the scope of this invention. One common method in toincubate the embryos in vitro for about 1-7 days, depending on thespecies, and then reimplant them into the surrogate host.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gamete or gametes. Thus, the gamete nuclei must beones which are naturally compatible, i.e., ones which result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is used. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material which canbe added to the nucleus of the zygote or to the genetic material whichforms a part of the zygote nucleus. If no genetic material is removed,then the amount of exogenous genetic material which can be added islimited by the amount which will be absorbed without being physicallydisruptive. Generally, the volume of exogenous genetic material insertedwill not exceed about 10 picoliters. The physical effects of additionmust not be so great as to physically destroy the viability of thezygote. The biological limit of the number and variety of DNA will varydepending upon the particular zygote and functions of the exogenousgenetic material and will be readily apparent to one skilled in the air,because the genetic material, including the exogenous genetic material,of the resulting zygote must be biologically capable of initiating andmaintaining the differentiation and development of the zygote into afunctional organism.

The number of copies of a transgene (e.g., the exogenous geneticmaterial or targeting vector constructs) which are added to the zygoteis dependent upon the total amount of exogenous genetic material addedand will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of atargeting vector construct, in order to insure that one copy isfunctional.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of an exogenous polynucleotide (e.g, that ofa targeting vector) by any suitable method as described herein.Alternative or additional methods include biochemical. assays such asenzyme and/or immunological assays, histological stains for particularmarker or enzyme activities, flow cytometric analysis, and the like.

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different knockout, or both. Alternatively, the partner may bea parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in hitro, orboth. Using either method, the progeny may be evaluated using methodsdescribed above, or other appropriate methods.

Retroviral infection can also be used to introduce a targeting vectorinto a non-human animal. The developing non-human embryo can be culturedin vitro to the blastocyst stage. During this time, the blastomeres canbe targets for retroviral infection (Jaenich, R. (1976) Proc. Nat. Acad.Sci. 73:1260-1264.) Efficient infection of the blastomeres is obtainedby enzymatic treatment to remove the zona pellucida (Manipulating theMouse Embryo, Hogan eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1986.) The viral vector system used to introduce thetargeting vector is typically a replication-defective retroviruscarrying the exogenous nucleic acid (Jahner et al. (1985) Proc. Nat.Acad. Sci. 82:6927-6931; Van der Putten et al. (1985) Proc. Nat. Acad.Sci. 82:6148-6152.) Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe targeting vector (e.g., the exogenous nucleic acids) sinceincorporation occurs only in a subset of the cells which formed thetransgenic non-human animal. Further, the founder may contain variousretroviral insertions of the transgene at different positions in thegenome which generally will segregate in the offspring. In addition, itis also possible to introduce transgenes into the germ line byintrauterine retroviral infection of the midgestation embryo (Jahner etal. (1982), supra.)

The cre-lox system, an approach based on the ability of transgenic mice,carrying the bacteriophage Cre gene, to promote recombination between,for example, 34 bp repeats termed loxP sites, allows ablation of a givengene in a tissue specific and a developmentally regulated manner (Orban,et al. (1992) Proc. Nat. Acad. Sci. 89:6861-6865.) LoxP sites can beplaced flanking an exon of any given gene. Thus, transgenic micecarrying the Cre gene under the control of a selected promoter can becrossed with transgenic mice carrying a transgene flanked by loxP sitesto generate doubly transgenic mice. The pioneering work in developingthis system was carried out by Orban et al. (1992) Proc. Nat. Acad. Sci.89:6861-6865. In one embodiment, the invention uses this technology totarget specific tissues in mice (e.g., expressing CMAH), in adevelopmentally regulated fashion in order to produce a mouse lackingNeu5Gc. This same method can easily be adapted for other mammalianspecies.

Gene targeting producing gene knock-outs allows one to assess in vivofunction of a gene which has been altered and used to replace a normalcopy and to generate knockout animals with utility as food. Themodifications include insertion of mutant stop codons, the deletion ofDNA sequences, or the inclusion of recombination elements (lox p sites)recognized by enzymes such as Cre recombinase. The Cre-lox system asused in one embodiment of the present invention allows for the ablationof a given gene or the ablation of a certain portion of the genesequence. The Cre-lox system was used to generate CMAH knockout miceexhibiting reduced. Neu5GC.

In another aspect, the invention relates to the use of a CMAH knockoutanimal, in particular an animal used for food stuff to generate food,food products, pharmaceuticals, and biologics for use.

In a further embodiment, the invention relates to cells and tissues thatcarry mutations in CMAH. The cells can be primary cells or establishedcell lines obtained from the transgenic animals of the inventionaccording to routine methods, i.e. by isolating and disintegratingtissue.

Such cells and tissues derived from the animals of the invention, inwhich the activity of CMAH has been reduced or abolished, are useful inin vitro methods relating to the study of sialic acid moieties, bindingand diseases and disorders related thereto.

Cells and cell lines derived from the knockout animals are furtheruseful in screening systems. The invention demonstrates that knockout ofCMAH results specific decreases of Neu5Gc. No obvious morphologicaldefects were noted in CMAH knockout mice.

Other Sources for Neu5Gc-Free Cells and Biological Medium.

In addition to producing Neu5Gc-free non-human mammalian productsutilizing transgenic techniques, many general cell culture techniquescan be used instead. As used herein, the term “devoid of Neu5Gc” intendsthat the ratio of Neu5Gc to total sialic acids is less than 5%, and evenless than 1%. For example, the immunogenicity of Neu5Gc can be exploitedby utilizing anti-Neu5Gc antibodies in an affinity column to remove anyNeu5Gc present in the products. Alternately, human cells of manyvarieties, tissues, embryoid bodies, neural lineage cells, carcinomacells, skin cells, and organs (collectively referred to herein as“cells”) can be cultured and preserved in Neu5Gc-environments, so longas they are free of Neu5Gc which can be incorporated therein and arerelatively free of anti-Neu5Gc antibodies, which would then bepotentially passed on to their human recipients. In another embodiment,serum replacements are now available to culture cells and tissues thatlack animal products altogether. Human orthologs and recombinantproteins lacking Neu5Gc are also suitable for incorporation into cellgrowth medium.

Besides HESCs, other cell types that are within the scope of the presentinvention include, for example, islet cells, endothelium, liver cells,kidney cells, cardiac cells, fibroblasts, etc. Most notably, however,progenitor cells and pluripotent cells that are not yet completelydifferentiated are of great use to scientific studies as potentialsources for therapeutic treatments involving cellular implantation. Inaddition, the methods described herein can be used to preserve humanorgans prior to transplation under conditions that avoid passing onanti-Neu5Gc antibodies or incorporation of additional Neu5Gc.

In most instances, cells are cultured on animal feeder layers, mostcommonly mouse fibroblasts. However, for reasons discussed elsewhereherein, these feeder layers only serve as additional sources forundesirable Neu5Gc, which can then be incorporated into the cells beingcultured. Accordingly, in an attempt to be overcautious, it may benecessary to completely eliminate Neu5Gc by using human serum in theculture medium with human feeder layers, ideally starting with freshHESCs that have never before been exposed to non-human mammals.

Both the lysosomal sialidase and the lysosomal sialic acid transporterare required for incorporation of glycoprotein-bound NeuGc into humancells. Inhibitors of such enzymes and transporter systems are known. Byincorporating these inhibitors, Neu5Gc uptake in cell and tissuecultures, as well as organs being preserved for transplantation can beeliminated.

EXAMPLES Example I Mechanism of Uptake and Incorporation of Neu5Gc intoHuman Cells

Experimental Procedures

Materials—Neu5Gc and Neu5Ac were purchased from Inalco Spa (Milano,Italy) and Pfanstiehl Laboratories, Inc. (Waukegan, Ill.)1,2-diamino-4,5-methylene dioxybenzene (DMB), chlorpromazine, gemstein,nystatin, amiloride, and saponin were purchased from Sigma-Aldrich (StLouis, Mo.) Premium Human Serum type AB was purchased from IrvineScientific (Santa Ana, Calif.) Neu5Ac aldolase was purchased from ICN(Costa Mesa, Calif.) All the reagents used were HPLC grade.

Cell Lines—Caco-2 cells (human epithelial cells isolated from a primarycolon carcinoma), normal human skin fibroblasts (CCD-919-SK) and chinesehamster ovary (CHO-K1) cells were purchased from ATCC (Manassas, Va.)Mutant human skin fibroblasts (GM05520, GM08496 and GM01718) wereobtained from the Coriell Institute for Medical Research (Camden, N.J.)Chimpanzee Fred and human LB EBV-transformed lymphoblasts were a giftfrom Dr. Peter Parham, Stanford University, CA.

Cell Culture—Caco-2 cells were propagated in alpha-MEM containingGLUTAMAX™ (Invitrogen, San Diego, Calif.) and a mixture ofribonucleosides and deoxyribonucleosides (Gibco/Invitrogen, San Diego,Calif.) supplemented with 20% FCS. All the fibroblast cell lines andCHO-K1 cells were cultured in the same media supplemented respectivelywith 15% non-heat inactivated FCS or 10% heat inactivated FCS.Chimpanzee Fred and human LB EBV-transformed lymphoblasts were culturedin RPMI-1640 (Gibco/Invitrogen, San Diego, Calif.) supplemented with 10%heat inactivated FCS or 15% human serum. All of the cultures weremaintained at 37° C., 5% CO₂ atmosphere. In order to deplete anyremaining Neu5Gc from FCS, the cells were split and cultured prior toNeu5Gc feeding experiments for at least 4 days in alpha-MEM supplementedwith an adequate percentage of heat-inactivated premium human seruminstead of FCS. The cells were then maintained under the same conditionsduring the whole feeding experiment. The human serum was heatinactivated at 56° C. for 30 min. before use.

Preparation of ManNGc from Neu5Gc—ManNGc was prepared by incubating 73μmoles of Neu5Gc with 624 U Lactate dehydrogenase, 30 μmoles NADH and 10U Neu5Ac Aldolase, EC 4.1.3.3, in 15 ml of 100 mM potassium phosphatebuffer, pH 7.2. The incubation was carried out at 37° C. for 16 h. TheManNGc was separated from any unreacted Neu5Gc by passing the productserially over AG50WX-2 and AGIX-8 (Bio-Rad, Richmond, Calif.)ion-exchange resins. The run-through and 5 column volumes of waterwashes were collected and concentrated by freeze-drying. The reactionyield (91-98%) was followed by the disappearance of Neu5Gc, using DMBderivatization of the reaction mixture and analysis by HPLC (asdescribed elsewhere herein.)

Preparation and Purification of Sia from Bovine Submaxillary Mucin—Amixture of standard Sias were prepared from bovine submaxillary mucin.Total mucins were extracted from frozen submaxillary glands using knownmethods. Sias then were released with mild acid, collected by dialysis(1000 daltons molecular-weight-cut-off) and purified on ion exchangecolumns under conditions determined to minimize loss of O-acetylation.

Neu5Gc and ManNGc Feeding Experiment—Neu5Ac, Neu5Gc or ManNGc weredried, dissolved in the appropriate media supplemented withheat-inactivated human serum, sterilized using a Spin-X® (Corning Inc.,Corning, N.Y.) and then added to the cells. The pH of the mediacontaining Sia was adjusted to neutrality using sterilized 1M NaOHbefore starting the feeding experiment. Cells were cultured in thepresence of up to 3 mM free Sia or ManNGc for 1 or 3 days at 37° C. Atthe end of the feeding, cells were washed with cold PBS, harvestedeither by scraping or with 2 mM EDTA for fibroblasts, and washed againwith cold PBS prior to fractionation.

Fractionation of the Labeled Cells—Washed cell pellets were sonicatedinto 500 μL of 20 mM sodium phosphate buffer or 20 mM Tris-HCl, pH 7.5,using 4×15 second pulses of a sonicator cell disrupter, model SomeDismembrator (Fisher Scientific, Hampton, N.J.) at a probe setting of 3.The sonicate was centrifuged at 75×g for 15 min., and the pelletObtained consisted primarily of nuclei and unbroken cells. The pelletcontained <5% of the incorporated sialic acid as determined using aradioactive tracer (data not shown.) The supernatant was thereforeconsidered as the “total homogenate” fraction. A portion (20%) of the“total homogenate” fraction was taken for protein quantification and Siaanalysis by DMB derivatization and HPLC analysis (as discussed below.)The remainder was centrifuged at 100,000×g for 1 h. The resultingpellet, called the “membrane” fraction, was then resuspended bysonication (15 sec.) in 200 μl of sodium acetate buffer, pH 5.5. The100,000×g supernatant, called the “soluble” fraction was adjusted to 90%ethanol using absolute ice-cold ethanol, and placed overnight at −20° C.The flocculant precipitate, which represents the “soluble protein”fraction, was washed 3 times with 90%® ice-cold ethanol and thenresuspended in 200 μl of water. The supernatant fluid representing thecytosolic low molecular weight (LMW) fraction was dried and brought upin 100 μL with water prior to Sia analysis. All the Sias in thesefractions were released with mild acid hydrolysis if necessary and thenanalyzed by HPLC after DMB derivatization. Protein quantification wasperformed on the total homogenate, membrane and soluble proteinfractions by using the BCA protein assay kit from Pierce (Rockford,Ill.) In some experiments, the resulting data obtained for the Sia boundto the membrane fraction and to the soluble proteins were pooled andpresented here as a High Molecular Weight (HMW) fraction.

Sialic Acid Release, DMB Derivatization and HPLC Analysis—The bound Siasfrom the total homogenate, membrane and soluble protein fractions werereleased using 2M acetic acid hydrolysis, 3 h. at 80° C. The releasedSias, or free Sias, contained in the soluble LMW fraction were passedthrough a Microcon® YM-10 filter (Millipore, Bedford, Mass.) prior toDMB derivatization, which was done according to Hara et al., 1989.DMB-Sia derivatives from the different fractions were then analyzed byHPLC using a C18 column (Microsorb MV-TM 100 A, Varian, Palo Alto,Calif.) Isocratic elution was achieved using 7% methanol, 8%acetonitrile in water during 50 min. at 0.9 ml/min flow. The eluant wasmonitored by fluorescence.

Quantification of Sias—For all HPLC chromatograms, the quantification ofSias was done by comparison with known quantities of DMB derivatizedNeu5Gc and Neu5Ac used as standards and then reported in terms of pmolesof Sia. For total homogenate, membrane and cytosolic protein fractions,this number was expressed per mg of protein. Due to minorsample-to-sample variations in amounts and recoveries, the data in theFigures is presented as percent of Neu5Gc over total Sias, rather thanas absolute amounts.

MS and MS/MS Analysis of DUB Derivatives—In some experiments, the natureof the DMB derivatives of Sias was confirmed by mass spectrometry on aFinnigan MAT HPLC (Thermo, Waltham, Mass.) with online mass spectrometrysystem using a model LCQ-Mass Spectrometer System A. A Varian C18 columnwas used and eluted in the isocratic mode with 8% acetonitrile, 7%methanol, 0.1% formic acid in water at 0.9 ml/min over 50 min. Theeluant was simultaneously monitored by UV absorbance at 373 nm and byelectrospray ionization (ESI) mass spectrometry. The ESI settings usedwere capillary temperature of 210° C., capillary voltage at 31 V and thelens offset voltage at 0 V. Spectra were acquired by scanning from m/z150-2000 in the positive ion mode. In some instances, MS/MS was acquiredby selecting the parent mass and using a 20% normalized collisionenergy. Data analysis was performed using the XCALIBUR data analysisprogram from the instrument manufacturer.

Endocytosis Inhibition Experiments—Caco-2 or normal fibroblast cellswere split and cultured in alpha-MEM media supplemented respectivelywith 20% or 15% human serum for 4 days before starting the endocytosisdrug inhibition experiments in order to deplete any Neu5Gc derived fromFCS. Cells were then pre-treated for 2 h. with the specific inhibitorsunder the same culture conditions. Fresh media containing the sameamount of inhibitor and 3 mM of Neu5Gc was then added to the cells,which were incubated for 16 h or 3 days and finally harvested andfractionated as described above. Based on known methods, chlorpromazine,genistein, nystatin and amiloride were used at final concentrations of6˜Lg/mL, 200 ItM, 25 pg/mL and 3 niM, respectively.

Western Blot Analysis—Membrane proteins extracted from Neu5Gc-fed,ManNGc-fed or non-fed human wild-type (WT) and mutant fibroblasts wereseparated by SDS-PAGE electrophoresis using an 8% polyacrylamide gel.The separated proteins were transferred onto a nitrocellulose membrane,which was blocked overnight with tris buffered saline containing 0.1% ofTween-20 (TBS-T.) Immunodetection was then performed using ananti-Neu5Gc antibody (1:10,000 in TBS-T, 3 h., room temperature (RT).)Binding of the anti-Neu5Gc antibody was detected using a secondary horseradish peroxidase (HRP)-conjugated donkey anti-chicken IgY antibodydiluted at 1:30,000 in TBS-T for 45 min. at room temperature (RT)(Jackson ImmunoResearch Laboratories, West Grove, Pa.) Final developmentof the blots was performed using Supersignal West Pico ECL reagent(Pierce, Rockford, Ill.) and X-GMAT Kodak (Rochester, N.Y.) films.

Flow Cytometry—Human WT and mutant fibroblasts grown in media with 10%FCS+20% horse serum for 3 days were lightly trypsinized (0.04% trypsin,0.53 mM EDTA for 5 min.) to release cells from the flasks. The cellswere washed with PBS and then fixed overnight with 1% paraformaldehydein PBS. Fixed cells were permeabilized or not with 0.1% saponin in PBSat RT for 20 min. Chicken anti-Neu5Gc antibody was added to cells at a1:200 dilution in PBS and incubated at RT for 30 min. Cells were thenwashed with PBS and resuspended in FITC-conjugated goat anti-chicken IgY(1 μg/100 μl) (Southern Biotechnology Associates, Birmingham, Ala.) andallowed to incubate for 30 min. at RT. Labeled cells were washed withPBS and resuspended in 500 μl PBS for analysis of FITC fluorescence on aFACS Calibur (BD Biosciences, San Jose, Calif.)

Fluorescence Microscopy—Human WT and mutant fibroblasts were grown onpoly-D-lysine-coated glass chamber slides (Nalge Nunc. International,Naperville, Ill.) with media containing 10% FCS+20% horse serum for 4days. Cells were fixed onto slides using 1% paraformaldehyde in PBS for30 min. at RT before permeabilizing with 0.1% saponin for 20 min. at RT.Chicken anti-Neu5Gc antibody was then added at 1:50 dilution in PBSalong with 1 μg of mouse anti-LAMP-1 (clone H4A3, BD Pharmingen, SanDiego, Calif.) and incubated at RT for 1 h. Bound antibodies were thendetected with FITC-goat anti-chicken IgY and Cascade Blue (Invitrogen,San Diego, Calif.)-goat anti-mouse IgG (each at 1 l·μg/100 μl) at RT for1 h. Cells were washed with PBS and covered with Gel/Mount (Biomedia,Foster City, Calif.) before fluorescence imaging with a Zeiss (CarlZeiss, Germany) microscope at 400× magnification with emission filtersat 400 and 520 nm for Cascade Blue and FITC, respectively.

Results

Free Neu5Gc can be taken up by human epithelial cells from an exogenoussource and incorporated into different subcellular fractions. Evidencewas presented suggesting that the small amounts of Neu5Gc found in somehuman tissues originated from dietary sources and showed that humanCaco-2 cells (human epithelial cells from a primary colon carcinoma) inculture could metabolically incorporate free Neu5Gc, as determined by aWestern blot of a total homogenate using and anti-Neu5Gc antibody.Increasing incorporation of Neu5Gc was found in the total homogenatefraction of the cells over time, with the highest level reached afterincubation with 3 mM Neu5Gc for 3 days. Moreover, western blotting withan anti-Neu5Gc antibody demonstrated metabolic incorporation of Neu5Gcinto glycoproteins of these cells.

The partitioning of the exogenous Neu5Gc into different subcellularfractions of these cells has also been analyzed. Prior to feeding,Caco-2 cells were split and cultured in human serum instead of FCS inorder to eliminate traces of Neu5Gc in the cells. Culture was continuedfor 3 days in the presence of 3 mM Neu5Gc using 3 mM ManNGc and Neu5Acas positive controls. Indeed, it was shown that Neu5Ac and ManNGc can beincorporated into cells and that ManNGc or its peracetylated form can bemetabolized into Neu5Gc. After the 3 day feeding, the cells wereharvested and the Neu5Gc content of the different subcellular fractionswere analyzed by DMB derivatization, HPLC, MS and MS/MS analysis. Asshown in FIG. 1A, the DMB-HPLC profiles of Sias released from themembranes of Caco-2 cells fed with 3 mM ManNGc or Neu5Gc presented twopeaks which correspond to Neu5Gc and Neu5Ac, by comparison with theretention times of standards. Cells that were not fed or fed with Neu5Achad only one peak corresponding to Neu5Ac. These results were confirmedby LC-MS and MS/MS analysis. DMB-Neu5Gc and DMB-Neu5Ac adducts gavesignals at m/z 442/424 and 426/408 respectively, representing molecularions of DMB-derivatized Neu5Gc and Neu5Ac and their dehydrated forms.LC-MS and MS/MS data obtained on DMB-derivatized Sias released from themembranes of Caco-2 cells non-fed or fed with Neu5Ac gave only a singleion at m/z 426 which can be broken down to 408 by MS/MS, confirming thepresence of Neu5Ac and the absence of Neu5Gc. The same analysis onNeu5Gc or ManNGc fed Caco-2 cell membrane Sias gave ions at m/z 442/426,which are respectively dehydrated to 424/408 in MS/MS analysis.

These analyses confirmed the presence of Neu5Gc associated specificallywithin the glycoconjugates of the membranes of Caco-2 cells fed withManNGc or Neu5Gc. All other sub-cellular fractions were also studiedusing the same DMB-HPLC approach. Due to sample-to-sample variations inamounts and recoveries, data is presented in this and subsequent figuresas percent of Neu5Gc over total Sias, rather than as absolute amounts.FIG. 1B summarizes the results showing that the total homogenate (TH),high molecular weight fraction (HMW is the combination of membrane andsoluble protein fractions) and cytosolic low molecular weight (LMW)fraction contain 58, 46 and 70% Neu5Gc, respectively. In the experimentpresented here, the % of Neu5Gc in the membrane fraction of cells fedwith Neu5Gc was lower compared to the one obtained for cells fed withManNGc. This was not always the case, as was observed in other feedingexperiments that free Neu5Gc can be as efficient as ManNGc and sometimeseven better. The relative percentages obtained for the other fractions(total homogenate, LMW and soluble protein) were similar in severalrepeated ManNGc and Neu5Gc feeding experiments.

The uptake mechanism of free Neu5Gc is not specific for human epithelialcells. The above experiments showed that free Neu5Gc can be taken up byhuman epithelial carcinoma cells from the media, and incorporated intodifferent subcellular fractions such as membrane-bound glycoconjugates,soluble proteins, and low molecular weight compounds present in thecytosol. To see if this is a specialized mechanism inhuman carcinomacells, similar Neu5Gc feeding experiments were done on other human celltypes such as normal skin fibroblasts and neuroblastomas. It was foundthat fibroblast cells can also take up free Neu5Gc from the media,albeit in a less efficient manner. As presented in FIG. 2A, 28%, 39% and41% Neu5Gc are present in the TH, HMW and cytosolic LMW fractions of thehuman normal fibroblasts after Neu5Gc feeding. Lower levels (4%, 19%,12% Neu5Gc) were already present in the same fractions when fibroblastcells were not incubated in presence of 3 mM Neu5Gc. This Neu5Gc isassumed to be derived from Neu5Gc on FCS glycoproteins used for cellculture, prior to feeding experiments Human neuroblastoma cells alsoincorporate Neu5Gc with an efficiency comparable to the Caco-2 cells(FIG. 2B.) These data indicate that the uptake mechanism of Neu5Gc canalso occur in other human cell types, with varying efficiencies.

The uptake mechanism of free Neu5Gc is also not specific for humancells. To determine if the uptake mechanism of free Neu5Gc is specificfor human cells, Neu5Gc feeding of human and chimpanzee lymphoblasts wascompared. Humans are evolutionarily most closely related to thechimpanzee, whose proteins are ˜99% identical to those of humans. Ofcourse, great apes such as chimpanzees are able to express Neu5Gc inlarge amounts because they have an active form of the CMP-Neu5Achydroxylase. Prior to the feeding experiment, both cell types were splitand cultured in human serum instead of FCS for a couple of weeks. Asexpected, the Neu5Gc content of chimpanzee lymphoblasts could not beeliminated completely because of the endogenous production of Neu5Gc.After a 3 day feeding of 3 mM Neu5Gc or Neu5Ac, the cells wereharvested, fractionated and the Neu5Gc content of the differentsubcellular fractions were analyzed. As shown in FIG. 3A, the humancells fed with 3 mM Neu5Gc contained 67% Neu5Gc in the TH fraction, 51%in the HMW fraction and 80% in the LMW cytosolic fraction. In contrast,the same cells had almost no detectable Neu5Gc when they were non-fed orfed with Neu5Ac (FIG. 3A.) With chimpanzee cells, we measured baselinelevels at 57% Neu5Gc in the TH fraction, 66% in the HMW fraction and 70%in the LMW fraction of non-fed cells (FIG. 3B), representing theendogenous production of Neu5Gc by these cells. When the chimpanzeelymphoblasts were fed with 3 mM Neu5Ac, the percentages of Neu5Gcpresent in the different fractions changed only minimally (FIG. 3B),presumably because of biosynthetic transformation of Neu5Ac to Neu5Gcoccurring at the sugar nucleotide level. When the chimpanzeelymphoblasts were fed with 3 mM Neu5Gc, an increase above the baselinelevels was observed to 70% for the TH fraction, 72% for the HMW fractionand 84% for the LMW fraction (FIG. 3B). Similar experiments have beendone with Chinese hamster ovary (CHO-K1) cells and with epithelial cellsisolated from a spontaneous tumor from a CMAI-I gene knock out mouse.These experiments gave similar results. However, since non-human cellsoften have large endogenous amounts of Neu5Gc, the consequences are moredramatic in human cells.

Free Neu5Ac and Neu5Gc are taken up and incorporated by the samepathways. From these data, it appears that Neu5Ac and Neu5Gc can betaken up by many kinds of cells from an exogenous source andincorporated into endogenous glycoconjugates. It has previously beendemonstrated that Neu5Gc and Neu5Ac are used interchangeably byessentially all of the steps leading to their final incorporation intoglycoconjugates. Higa, H. H. et al. ((1985) J. Biol. Chem.260:8838-8849) showed that CMP-Sia synthetases from calf brain and frombovine and equine submaxillary glands both converted Neu5Ac and Neu5Gcto their CMP derivatives efficiently. They also studied six mammaliansialyltransferases purified from porcine, rat, and bovine tissues andconcluded that CMP-NeuAc and CMP-NeuGc were equally good donorsubstrates for all the enzymes. Schauer, R. et al. ((1980)Hoppe-Seyler's Z. Physiol. Chem. 361:641-648) showed that the frog liverCMP-Sia synthetases had very similar Km values for Neu5Ac and Neu5Gc. Ithas also previously been shown that CMP-Neu5Gc and CMP-Neu5Ac could betaken up by Golgi vesicles and incorporated into endogenousglycoproteins at an approximately equal rate. Similar observations weremade by Lepers, A. et al. ((1989) FEBS Lett. 250:245-250) in rat andmouse liver Golgi. Thus, by doing competition experiments in Caco-2 andhuman normal fibroblast cells, it can be determined whether Neu5Gc andNeu5Ac are taken up and incorporated via the same pathways. Both celllines gave similar results, and only the results for Caco-2 cells arepresented in FIG. 4. Feeding was done for 3 days with 3 mM Neu5Gc in theabsence or presence (3 mM or 15 mM) of Neu5Ac in the media. The baselineincorporation of 56% Neu5Gc in the TH was reduced to 48% in the presenceof 3 mM Neu5Ac and. further decreased to 35% in the presence of added 15mM Neu5Ac (FIG. 4A). The percentage of Neu5Gc was even more affected inthe membrane-bound fraction, reducing from 41% to 29.9% with 3 mMNeu5Ac, and almost to zero in the presence of 15 mM Neu5Ac (FIG. 4B).Since a 5-fold excess of Neu5Ac was enough to abolish the incorporationof Neu5Gc into the membrane fraction of the cells, it is concluded thatboth molecules likely use the same pathways to enter into human cellsand become available for metabolic incorporation. It is of coursepossible that there are minor differences in utilization of Neu5Gc andNeu5Ac by various enzymes and transporters in the pathways.

Free Neu5Gc enters into cells via pathways of endocytosis. Negativelycharged hydrophilic molecules like sialic acids usually do not crossmembranes. To understand how free Neu5Gc enters into cells, thehypothesis that it does so via endocytic pathways was explored. Thus,Neu5Gc feeding experiments on Caco-2 cells were done in the presence ofdrugs that are known to inhibit various endocytic pathways common tomost cell types. Based on known studies in the field, it was decided touse chlorpromazine for blocking the clathrin dependent pathway andnystatin and genistein for the clathrin independent pathways (with anadditional specific action of nystatin on caveolar uptake.) Amiloridewas used as an inhibitor of fluid phase pinocytosis. All of these drugswere used at concentrations based on prior studies. As before, theCaco-2 cells were incubated in an appropriate media containing humanserum instead of FCS and pre-treated with the drug for 2 hours, followedby the addition of 3 mM of Neu5Gc for 16 h. or 3 days. As shown in FIG.5A, incorporation of Neu5Gc in the TH fraction in the presence ofchlorpromazine and nystatin (˜65% in both cases) was about the same asfor the non-treated Caco-2 cells. In contrast, Neu5Gc incorporation intocells was decreased in the presence of genistein (51%) and much furtherby amiloride (35.4% Neu5Gc.) Analysis of incorporation intomembrane-bound glycoconjugates gave similar results. While there was noobvious difference in the Neu5Gc incorporation for cells culturedwithout (45%) or with chlorpromazine (50%) or with nystatin (44%),genistein and amiloride caused marked reduction of incorporation to 34%and 10% respectively. These results indicate that exogenous fine Neu5Gcenters cells via clathrin-independent endocytic pathways with a majorcontribution from fluid phase pinocytosis.

The lysosomal sialic acid transporter is required for export of freeNeuGe from the lysosome to tile Cytosol. Free Neu5Gc molecules enteringthe cell via endocytic pathways would still be restricted from passivelydiffusing out of endosomes into the cytosol. It was hypothesized thatthey would eventually reach the lysosome where they would have theopportunity to utilize the previously known lysosomal sialic acidtransporter (58-60) to reach the cytosol. To test this hypothesis,fibroblasts from a patient (GM05520) with a severe infantile form ofsialic acid storage disease (ISSD), a disease that is caused by agenetic defect in this transporter, were used. As shown in FIG. 6, thepercent of Neu5Gc incorporation into membrane-bound glyconconjugates wasreduced from 37% in normal wild-type (WT) fibroblasts to 5% in thesemutant cells. As a control, the metabolic conversion of ManNGc intoNeu5Gc in these cells was also studied, which presumably occursfollowing passive diffusion through the plasma membrane, and does notrequire the lysosomal sialic acid transporter. As predicted, it wasfound that there was essentially no difference in between normal (19%Neu5Gc) versus mutant fibroblasts (18% Neu5Gc) following feeding with 3mM ManNGc. Another similar mutant human fibroblast cell line (GM08496)was studied, with a partial inhibition of function of the lysosomalsialic acid transporter. This cell line was isolated from a patientsuffering from Salla disease, a milder adult form of sialic acid storagedisease. Neu5Gc feeding of these cells resulted in 16% Neu5Gc inmembrane-bound glycoconjugates in comparison to the 37% seen in normalWT fibroblasts. Again, feeding with ManNGc gave no obvious change fromthe control (17% Neu5Gc.) To further confirm that there was a differencein incorporation into glycoproteins, a Western blot analysis was carriedout of proteins, extracted from the membranes of wild-type and GM05520mutant human fibroblasts using an anti-Neu5Gc antibody, with or withoutprior Neu5Gc or ManNGc feeding. The mutant fibroblasts could notincorporate Neu5Gc into glycoproteins, but could in fact convert it fromManNGc (data not shown.) Taken together, the data confirm the hypothesisthat the lysosomal sialic acid transporter plays a crucial role indelivering free sialic acids that enter into cells via endocytosis tothe cytosol for activation and incorporation into glycoconjugates.

Both the lysosomal sialidase and the lysosomal sialic acid transporterare required for incorporation of glycoprotein-bound NeuGc into Humancells. Several studies have shown that when human cells are transferredfrom conventional media containing FCS into serum-free media or humanserum, the small amounts of endogenous Neu5Gc in these cells graduallydisappear. It has always been assumed that this is because FCS containsmany glycoproteins with attached Neu5Gc. However, the pathway by whichthese glycosidically-bound Neu5Gc molecules enter the cell andeventually become incorporated into endogenous glycoproteins has neverbeen defined. This question is also of direct relevance to human gutepithelial cells, which would be exposed to glycoprotein-bound Neu5Gc ofdietary origin (red meat, milk products for example.) Based on the abovefindings, it is reasonable to hypothesize that the Neu5Gc carrying serumglycoproteins enter the cell via fluid phase pinocytosis, eventuallyreaching the lysosome where they are exposed to the lysosomal sialidase.The resulting free Neu5Gc in the lysosome would then have theopportunity to use the lysosomal sialic acid transporter to reach thecytosol in order to be salvaged and eventually converted to CMP-Neu5Gc.

To test this hypothesis, the GM05520 mutant human fibroblasts, which arecompletely deficient in the lysosomal sialic acid transporter, as wellas GM01718 mutant human fibroblasts, which have less than 1% lysosomalsialidase activity compared to normal fibroblasts, were used. For thesestudies, it was important to differentiate between cell surface andinternal Neu5Gc. Thus, instead of subcellular fractionation, the methodof flow cytometry was utilized, using affinity purified Neu5Gc-specificchicken antibody. As shown in FIG. 7A, after 3 days of feeding with 10%FCS+20% horse serum (both rich sources of glycoprotein-bound Neu5Gc),the total surface expression of Neu5Gc was significantly lower in bothmutant fibroblasts compared to WT fibroblasts. Permeabilization of cellsrevealed similar levels of total Neu5Gc glycoconjugates (FIG. 7A), butthe majority in the two mutants was internal (FIG. 7B). To confirmtrapping of Neu5Gc glycoconjugates in lysosomes, fluorescence microscopyanalysis was performed of permeabilized fibroblasts, co-labelling cellswith a known marker for lysosomes, LAMP-1. An even distribution ofNeu5Gc staining on WT normal fibroblasts with little co-localizationwith lysosomes was found. On the other hand, both the lysosomalsialidase and the transporter mutants demonstrated significantaccumulation of Neu5Gc glycoconjugates in the lysosomes.

The results with the sialidase-deficient fibroblasts confirm thehypothesis that this enzyme must act to release free Neu5Gc fromglycoproteins and to make it available for metabolic incorporation. Theaccumulation of Neu5Gc glycoconjugates in the lysosomal transportermutant was unexpected. A likely explanation is that accumulation of freeSia at a high concentration in the lysosomes inhibits the action of thelysosomal sialidase, resulting in accumulation of glycosidically-boundNeu5Gc. The residual levels of Neu5Gc detected on the surface of bothmutant cells might be explained by direct incorporation of gangliosidesand GPI-anchored proteins bearing Neu5Gc from the serum. Taken together,these data indicate that bound Neu5Gc molecules that enter into humancells via pinocytosis are released by the lysosomal sialidase and arethen transported by the lysosomal sialic acid transporter to thecytosol, where they are available for activation and incorporated intoglycoconjugates (FIG. 8.) Of course, depending on the type ofglycoprotein involved, bound Neu5Gc could also be delivered to lysosomesvia other pathways of endocytosis, e.g., receptor-mediated endocytosisvia clathrin-coated vesicles.

It has long been assumed that free sialic acids could not be efficientlyincorporated into cells because of their negative charge and hydrophilicnature. Thus, neutral ManNAc has traditionally been used as a precursorto feed cells for conversion into Neu5Ac. The same concept has beenapplied to various unnatural mannosamine derivatives, and the additionof O-acetyl esters to the hydroxyl groups of mannosamine derivatives hasbeen used to enhance delivery across the plasma membrane. In fact, oneearly study suggested that radioactive sialic acids could beincorporated into cells, and more recent work of others has shown“efficient” uptake of a variety of kinds of sialic acids into cells.However, the kinetics of incorporation showed no evidence of saturationeven at >10 mM concentrations, suggesting that the uptake was not due toa high efficiency cell surface transporter for sialic acids. Studiesusing a natural sialic acid (Neu5Gc) gave similar results.

It has been shown that sialic acids from the medium can be taken up intocells via non-clathrin-mediated mechanisms, mostly amiloride-sensitivefluid-phase pinocytosis. The content of the resulting pinocytoticvesicles and endosomes would eventually be delivered to the lysosome,where the previously described sialic acid transporter then delivers themolecules into the cytosol. It has also been shown that theincorporation of glycosidically-bound Neu5Gc from exogenousglycoproteins occurs by similar delivery to the lysosome, and release bythe lysosomal sialidase, followed by export into the cytosol (FIGS. 7and 8.) Once activated to CMP-Neu5Gc, molecules from both sources (freeand originally bound) would be indistinguishable from those that wereendogenously synthesized by the cells.

Most recently, it has been shown that human embryonic stem cells canincorporate Neu5Gc from medium glycoconjugates, making them targets forthe naturally occurring antibodies that circulate in most humans.Preliminary data also suggest that these antibodies could also berelated to diseases in intact humans. Thus, the mechanism by whichNeu5Gc is incorporated into human cells is of potentially greatimportance. Further studies of this process are also relevant both tothe ongoing attempts by various groups to incorporate different kinds ofunnatural sialic acids into cultured cells, and also to efforts tounderstand how exogenous dietary Neu5Gc gain entry into normal humantissues. In this regard, it is of note that Neu5Gc accumulation appearsto be enhanced in naturally occurring tumors, and in fetal tissues. Itis suggested that this may be explained by the fact that fluid phasemacropinocytosis is enhanced by growth factors, which are expected to bevery prominent in these two situations.

Finally, the studies described herein demonstrate, perhaps for the firsttime, that an extracellular small molecule that cannot cross the plasmamembrane is delivered efficiently to the cytosol utilizing fluidpinocytosis and a specific lysosomal transporter. This approach couldthus potentially be generalized to any small molecule that has aspecific lysosomal transporter, but not a plasma membrane transporter.For example, one could envisage that the neutral sugars GlcNAc andGalNAc, which do not have a high efficiency plasma membrane transporter,could nevertheless be delivered to the cytosol via the lysosomalGlcNAc/GalNAc transporter. The prediction is that adding millimolarconcentrations of these sugars into the medium would result insignificant delivery to the cytosol.

Example II Human Embryonic Stem Cells Express an Immunogenic NonhumanSialic Acid

The Hl ES cell line (WiCell Research Institute, Inc., Madison, Wis.)cells were cultured on mitotically inactivated (mitomycin C treated)mouse embryonic fibroblasts (MEF, Specialty Media, Phillipsbuurg, N.J.)in DMEMJF12 Glutamax (Gibco), 20% “knockout” serum replacement (Gibco)or pooled human blood-type AB serum (Pel-Freeze, Rogers, Ark.), 0.1 in Mnon-essential aminoacids (Gibco), 0.1 mM betamercaptoethanol (Gibco),and 4 ng/mL βFGF-2 (R&D Systems, Minneapolis, Minn.). For EB culture, HlES cells were grown in suspension for 7-10 days, using the same mediumwithout FGF-2 and 10% serum. Cells were changed to a new dish every dayto eliminate eventual fibroblast contamination.

HESC Transfection—Hl HESC were stably transfected to express greenfluorescent protein (GFP) by CAG-EGFP SIN lentivirus infection. The SINlentiviral vector expressing EGFP under control of the CAG promoter wasderived from a multiply attenuated HIV vector system, but included a U3deletion and introduction of a cPPT element. Vectors were produced bytriple transduction of HEK 293 cells (Graham et al., J. Gen Virol(1977), 36 (1):59-74) followed by ultracentrifugation and titration.Undifferentiated cells were exposed to the virus at a titer of 0.5×10¹⁰gtu/mL for 1 hour followed by a 2 day recovery period. EGFP was detectedby native fluorescence at day 3 after transduction. Cells expressingEGFP were FACS sorted for uniform EGFP expression. No loss in EGFPexpression was observed during propagation or EB differentiation and upto 10 months after transduction. The EGFP positive cells derived fromthese colonies are thus polyclonal in origin. The GFP positive ES cellsmaintain a similar phenotype to the wild type cells (SSEA-4, SSEA-3 andOct4-positive.)

Oct-4 antibody (1:500) was from Santa Cruz (Santa Cruz, Calif.), theother marker antibodies (SSEA-3, TRA-1-60, alkaline phosphatase andnestin) were from Chemicon (Temecula, Calif.; dilution 1:100), and thesecondary Cy3 antibody from Sigma (San Louis, Mo.; dilution 1:250).Alkaline Phosphatase (AP) activity was measured using the Vector RedAlkaline Phosphatase substrate kit I from Vector laboratories(Burlingame, Calif.)

Human sera—Sera from several healthy human donors were obtained afterwritten consent and Institutional Review Board approval, and anonymouslynumbered before further use. Anti-Neu5Gc antibody levels in severalserum samples were determined using known methods'. Two specific sera,corresponding to the lowest and highest extremes of the range, wereselected for the experiments. Another serum with a high level ofanti-Neu5Gc antibodies was also studied with identical results to thosepresented in the figures.

Determination of Neu5Gc content—Sias from HESC, feeder layer cells, EBor culture medium were released by mild acid, derivatized with1,2-diamino-4,5-methylene dioxybenzene (DMB) and analyzed by HPLC todetermine the percentage of Neu5Gc in total Sias.

Flow cytometry—Cells were harvested into 2 mM EDTA in phosphate buffer(PBS) and washed with PBS. 1×10⁵ cells were incubated with a chickenanti-Neu5Gc (1.5 μg/100 μL) and stained with a donkey anti-chicken IgYconjugated to Cy5 (Jackson, West Grove, Pa.; dilution 1:100 in PBS.)Neu5Gc-specific antibody binding was partially blocked by co-incubationwith 1% chimpanzee serum, which (unlike human serum) is rich in Neu5Gc.

For human serum antibody deposition studies, HESC were harvested andexposed to individual human sera. Human IgGs deposited on the cells werestained with an anti-human IgG conjugated to Alexa 594 (MolecularProbes, Carlsbad, Calif.; dilution 1:100 in PBS.) For C3b deposition,HESC were exposed to human serum, then incubated with a goat anti-humanC3b (Fitzgerald, Concord, Mass.; dilution 1:100 in PBS) and finallystained with an anti-goat I-G conjugated to Alexa 594 as above.

Cytotoxicity assays—A standard procedure for testing antibody,complement-mediated cytotoxicity after exposure to human sera, wasfollowed. HESCs were harvested and resuspended into GVB²⁺ buffer (Sigma)alone (control) or GBV²⁺ containing 25% human serum. Cells wereincubated for 2 h. at 37° C. and gently shaken. Dead cells were stainedwith propidium iodide (5 μg/mL) and analyzed by FACS. For cytotoxicityassays on the plate, cells were exposed to serum-free HESC culturemedium containing 25% of the test human sera. After 30 minutes at 37°C., they were harvested and stained with propidium iodide.

Statistical analysis—Sia content from at least two experiments run induplicate was analyzed using the T test in Microsoft Excel. Data areexpressed as mean±standard deviation.

Presence of Neu5Gc on HESC grown under standard conditions—Neu5Gc onHESC was detected using an affinity-purified chicken polyclonalmonospecific anti-Neu5Gc antibody. HESC stably expressing EGFP weregated for EGFP-positivity to separate them from contaminating feederlayer fibroblasts. The antibody stained HESCs growing in standardconditions, and binding was partially blocked by Neu5Gc-containingglycoproteins from chimpanzee serum (FIG. 9 a.) Blocking was incomplete,likely because not all possible epitopes recognized by the polyclonalantibody are present in chimpanzee serum.

To chemically analyze the Sia content of HESCs, they were separated fromthe feeder layer fibroblasts by FACS sorting using their EGFP signal.Feeder-layer-free EB derived from HESC were also examined withoutsorting. Both the membrane and cytosolic fractions from HESC and EB hada peak corresponding to Neu5Gc (FIG. 9 b), whose identity was confirmedby electrospray mass spectrometry (data not shown). HESC membranescontained 17.88±1.47 pmoles Sia/μg protein with 9.31±3.70 pmoles Sia/μgprotein in the cytosolic fraction. The percentage of total Sias presentas Neu5Gc varied from 6-10.5% in the membranes and from 2.5-9% in thecytosolic fraction. EB membranes had 16.59±3.88 pmoles Sia/μg proteinwith 9.13±0.10 pmoles Sia/μg protein in the cytosolic fraction. Thepercentage of total Sias present as Neu5Gc in EB varied from 5-17% forthe membranes and 6.5-11% for the cytosolic fraction.

Identifying potential sources of Neu5Gc in HESC—Since human cells areunable to synthesize Neu5Gc, the Neu5Gc detected likely originated fromelsewhere, eventually being metabolically incorporated by the HESC. Asexpected for other mammals, Neu5Gc represented 20% of total Sias in themouse feeder layer (0.92±0.13 nmoles/million cells.) However, uptakefrom feeder cells cannot explain all the Neu5Gc found in HESC, sinceremoval of the layer to obtain EB did not eliminate it. It was shownthat human cells can take up Neu5Gc from the medium and metabolicallyincorporate it into membrane glycoconjugates. The “serum replacement”containing medium used to support HESC growth was found to contain 35.93nmoles Neu5Gc/mL, representing 54% of total Sias. The commercial“knockout” serum replacement used for preparing this medium is the majorsource of Neu5Gc, since it contains 129 nmoles/mL. In contrast, mediumwithout any additives is poor in Neu5Ge (0.008 nmoles/mL.)

Neu5Gc content of HESC is reduced by growth in heat-inactivated humanserum with low anti-Neu5Gc antibodies—Culture in heat-inactivated poolednormal human serum could markedly reduce Neu5Gc in human colon carcinomacells, apparently due to metabolic replacement by Neu5Ac in the humanserum. HESC was therefore incubated in medium containing heatinactivated human serum instead of the standard serum replacement (anapproach already suggested by others for different reasons.) First, alot of pooled human serum was screened and defined in which naturalanti-Neu5Gc antibodies were very low (hereafter called NHS.) In case anyresidual antibodies were active, heat inactivation was used to eliminatecomplement. HESC incubated in such NHS remained undifferentiated on thefeeder layer, expressing typical levels of markers ofnon-differentiation (alkaline phosphatase, Oct-4, SSEA-3, SSEA-4,TRA-1-60, and TRA-1-81 and lack of all differentiation markers tested(FIG. 10.) After feeder layer removal, these HESC were able to developinto normal EB.

Neu5Gc incorporation into HESC membranes dropped after 3 days (from ˜4pmoles/μg protein to 0.34±0.06 pmoles/μg protein) and down to 0.13±0.01pmoles/μg protein after one week (−1% of total Sia as Neu5Gc, see FIG.11.) The required presence of the mouse feeder layer apparentlyprevented complete elimination of Neu5Gc from HESC. After growing for 3days in human serum, some HESC were differentiated into EB either in 10%commercial serum-replacement, or in 10% NHS, without a feeder layer.After one week in serum-replacement, the amount of Neu5Gc on the EBmembranes increased (from 0.34±0.06 to 0.40±0.10 pmoles/μg protein.) Incontrast, continued incubation in NHS further reduced Neu5Gc levels to0.047±0.06 pmoles/μg protein.

Natural human anti-Ncu5Gc antibodies bind to HESC and cause complementdeposition—Healthy humans have variable levels of “natural” circulatinganti-Neu5Gc antibodies. It was determined whether such antibodies couldrecognize Neu5Gc-containing epitopes on HESC grown under standardconditions. Cells exposed to a high-level anti-Neu5Gcantibody-containing human serum (Hi-GcAbHS) showed human IgG binding(FIG. 12 a shows only the EGFP+HESC.) In contrast, staining of cellsexposed to a low-level antiNeu5Gc antibody-containing human serum(Lo-GcAbHS) was similar to that of nonexposed controls. Antibodydeposition was related to the amount of Neu5Gc on the HESC, since cellsgrowing in NHS did not show any IgG binding when exposed to the sameHiGcAbHS (FIG. 12 b.)

Cell surface antibody deposition can activate the classical complementpathway, eventually leading to killing or phagocytosis. It wasdetermined whether complement C3b deposition occurred following exposureto Hi-GcAbHS. As before, the data was gated for EGFP+HESC. When HESCwere grown under the standard conditions, 37% were positive for C3b(FIG. 13 c; compare to 0% background in FIG. 13 a.) Only 22% of thecells were positive after exposure to Lo-GcAbHS, with actual levels onindividual cells being much lower (FIG. 13 b.) (Note that the Y-axis isa log scale.) When HESCs were grown for 5 days in NHS-containing medium,C3b-positivity after exposure to Hi-GcAbHS dropped to 13% (FIG. 13 c.)These data are consistent with deposition of anti-human IgG under thesame conditions (FIG. 12) and also with the significant reduction inNeu5Gc on the HESC after incubation in human serum-containing medium(FIG. 11.)

Such binding of antibody and complement to HESC would target them fordeath in vivo, via recognition by macrophages and NK cells. Regardless,attempts were made to directly determine antibody:complement-mediatedcytolysis on HESCs in vitro. The standard single cell suspensionrequired for such analyses caused extensive cell death even undercontrol conditions (without serum.) Exposure to Hi-GcAbHS causedincreased death above background levels seen with NHS, from 40% to60-70%. In contrast, the percentage of dead HESCs after exposure toLo-GcAbHS was similar to that of the control. When the assay wasperformed directly on the culture dish for shorter time, more HESCsremained alive. Cell death with Hi-GcAbHS was higher than that of thecontrol (14% vs. 10%), whereas the death rate after exposure toLo-GcAbHS remained unchanged.

HESCs and EB can incorporate the non-human Sia Neu5Gc from the murinefeeder layer and/or the medium, leading to an immune response mediatedby “natural” anti-Neu5Gc antibodies present in most humans. In effect,HESCs appear like animal cells to the human immune system. Pooled,heat-inactivated human serum selected for low titers of anti-Neu5Gcantibodies could be substituted for the traditional animal serum orserum replacement, supporting the undifferentiated growth of HESCs. Thisapproach markedly reduced the immune response, by reducing the Neu5Gccontent on the HESCs.

Most existing HESC lines have been grown or derived with mouse feederlayer. Standard culture conditions also include animal serum, or a serumreplacement. It is shown here that the commercial “serum replacement” isalso a rich source of Neu5Gc, and both HESCs and EB are able toincorporate it. The composition of this serum replacement is describedin PCT WO 98/30679, and includes proteins like transferrin, which arelikely to be from animal sources and therefore, would carry Neu5Gc.Human orthologs or recombinant proteins synthesized in bacteria could beused instead.

Many efforts have been recently made to eliminate these animal-derivedcomponents. The use of a feeder-free system, such as Matrigel or othercomponents of the extracellular matrices, have been explored. However,feeder-free conditions seem to facilitate in vitro evolution of HESCs,selecting for aneuploid cells. Moreover, many of the medium and matrixcomponents are still from animal sources and contamination with Neu5Gccan be expected.

Human feeders of different origins have also been tried. Richards et al.first reported successful derivation and culture of some HESC lines inthe complete absence of non-human components, using feeder layers fromhuman tissues with human serum and supplements, further demonstratingthe ability to develop teratomas, i.e., confirming the maintenance ofpluripotentiality. It was also noted that human serum did not cause anychange in the undifferentiated state of the HESCs. Others have alsotried similar xeno-free techniques on hematopoietic stem cells bygrowing them on human stromal cells and using medium containing human ABserum. Of course, the use of an “all-human” environment carries adifferent set of risks (unexpected contamination with novel or newlyemerging pathogens).

There are also potential implications for the incorporation of Neu5Gcwith regard to general HESC biology. Many characteristic markers ofHESC(SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) are glycolipids orglycoproteins, many of which can carry Sias. SSEA-4, which is highlyexpressed even in long-term cultures of HESCs, is the sialylated form ofthe globo-series glycolipid SSEA-3 (Gb5.) TRA-1-60 is a sialylatedkeratan sulfate protein and the TRA-1-81 epitope became accessible onlyafter sialidase treatment. Neural lineage cells derived from EB expressthe polysialylated form of NCAM, as well as the antigen A2B5, whichcorresponds to polysialylated gangliosides. Since Sias are involved inself-recognition events, the presence of Neu5Gc instead of Neu5Ac couldlead to unexpected impairments of cell function and tissue development.

Another possible solution is growth in heat-inactivated serum from theactual patient who is going to receive the therapy. Similar alternativeshave been suggested for hematopoietic stem cells. Even if the patientserum contains anti-Neu5Gc antibodies, heat inactivation could preventcomplement activation, until such time as the pre-existing Neu5Gc in theHESCs is metabolically eliminated by the Neu5Ac in the serum. An addedadvantage to this approach is that it would screen for allogeneiccytotoxic antibodies in the recipient's serum.

Example III Elimination of N-Glycolylneuraminic Acid Hydroxylase in aMouse Model

Material and Methods

Generation of targeting construct pFlox-Ex-SL for elimination of CMAHexpression. A 10 kb genomic DNA region spanning the CMAH gene thatincludes the 92 bp corresponding to exon 6 of the murine CMAH wasisolated from a BAC clone by digesting the clone with EcoRI withsubsequent Southern Blotting using a radiolabeled probe corresponding toexon 6. The 10 kb piece was subcloned into pBluescript II KS+,generating the construct pBS-35. Mapping of the restriction sites of the10 kb piece was performed by digesting pBS-35 with various restrictionenzymes. A 535 bp piece containing exon 6 was isolated from pBS-35 bydigesting with NheI and XbaI, and cloned into the BamHI site in thepFlox vector. A 1150 bp intronic region directly upstream of the 535 bppiece was isolated by digesting with NheI and subcloning into thepBluescript II KS+ vector. This was then used for PCR using forwardprimer CGGCTCGAGTGAGCTACATGAGAT and reverse primerGGGCTCGAGTAATCACCAAGCAAA, thereby adding XhoI restriction site the endsof the 1150 bp piece. The PCR product was subcloned into pBluescript IIKS+ vector, which was then digested with XhoI and cloned into the Xhosite in the pFlox-Exon vector, creating the pFlox-Ex-S vector. Next, a4850 bp piece directly downstream of the 535 bp piece was excise frompBS-35 by digesting with XbaI and NheI and cloned into the XbaI site inpFlox-Ex-S vector, generating the final targeting construct,pFlox-Ex-SL.

Generation of CMAH null mice. pFlox-Ex-SL plasmid DNA was purified by astandard cesium chloride method and linearized by digestion withrestriction enzyme Not I. The solution containing digested plasmid DNAis then subjected to sequential phenol, 1:1 phenol:chloroform, andchloroform extraction. The aqueous phase containing the linearized DNAis then subjected to sodium acetate precipitation. The resulting DNApellet is washed 2 times in ice-cold 70% ethanol, air dried, andresuspended in TE. The generation of the transgenic mice was performedby the UCSD Transgenic Mouse Core. In brief, the linearized transgenicconstructs were electroporated into embryonic stem cells (ES cells)isolated from the 129/SvJ mouse strain. The ES cells then underwent drugselection, subclone isolation, and growth of isolated clones. Each clonewas grown in triplicate plates, one that was kept by the Core as amaster plate that was frozen at −80° C. and two that were returned toinvestigators for the identification of homologous recombinants. DNA waspurified from each clone and subjected to screening by PCR and SouthernBlot analysis as described below. Homologous recombinants were thawed,expanded, and reconfirmed by PCR and Southern Blot analysis. For thegeneration of the CMAH null mouse, homologous recombinant clones weresubjected to transfection with Cre-recombinase expression vector,underwent gancyclovir drug selection against the presence of thymidinekinase (TK), subclone isolation, and growth of isolated clones. Thedesired type of recombination was then identified by PCR analysis.Karyotyping was then performed and two of the best clones were selectedfor blastocyst injection. Chimeric mice were then generated and bred toC57Bl/6 females to allow germline transmission of the transgene.

PCR genotyping analysis of CMAH null mice. To genotype the mice, DNAisolated from toe clips were used for PCR analysis. Toe clips performedto mark the identity of the mice were collected and digested in 20 ul ofbuffer containing 50 mM Tris, pH 8.0, 20 mM NaCl, 1 mM EDTA, 1% SDS, and250 ug/ml Proteinase K at 55° C. until the soft tissue dissolved. Thesample was then diluted with 180 ul of water and boiled to inactivatethe enzyme. For genotyping of CMAH null mice, PCR primers UpExon6(CCAGGAGGAGTTACCCTGAA), Exon6#2 (TCAATCAATTGCATGGGTCT), and DwExon6(CGAGGACAGCCCAGAGACTA) were designed based on the published murine CMAHsequence. Analysis was performed using the following PCR cycle: 94° C.for 5 min; 40 cycles of 94° C. for 30 sec, 53° C. for 30 sec, and 72° C.for 1 min; and 72° C. for 5 min. A PCR product of 305 bp is generatedfrom the deletion allele while a product of 490 bp is generated from thewild-type allele.

Southern blot analysis of the CMAH null mice. To genotype the mice, DNAisolated from tail clips were analyzed by PCR. Tail clips were digestedovernight at 55° C. in buffer containing 10 mM Tris-HCl, pH 8.0+1 mMEDTA (TE), 1% SDS, 140 mM NaCl, and 0.3 mg/ml Proteinase K. A solutionof TE, 5.3M NaCl, and chloroform was the added. The genomic DNA in theupper aqueous phase was subjected to ethanol precipitation andresuspended in TE.

DMB-HPLC analysis of Neu5Gc content in cells and tissues. Cells ortissues were homogenized and subjected to acid hydrolysis using 2Macetic acid at 80° C. for 3 h to release sialic acids from cellularglycoconjugates. After centrifugation at 20,000 g, the supernatant wasfiltered through a Microcon 10 unit, dried down, and reconstituted inwater. Aliquots were derivatized with 1,2-diamino-4,5-methylenedioxybenzene (DMB) and analyzed by HPLC (DMB-HPLC). To remove O-acetylesters, samples were incubated with 0.1 M NaOH for 30 min at roomtemperature to remove base-labile O-acetyl esters.

Immunohistochemistry using the anti-Neu5Gc antibody. Tissues werecollected from autopsies or unused pathological material frozen in OCTcompound and archived at −70° C. Frozen tissue sections were air-driedfor 30 min, fixed in 10% buffered formalin for 30 min, endogenousperoxidase activity quenched and non-specific binding sites blocked with5% (Neu5Gc free) human serum in PBS for 30 min. Sections were thenincubated with the anti-Neu5Gc antibody in 5% human serum/PBS at a 1:200dilution at RT for 2 h. After washing, HRP-conjugated donkeyanti-chicken IgY antibody in 5% human serum/PBS at a 1:100 dilution wasapplied for 1 hr. Control sections were incubated with secondary reagentonly or a control chicken IgY antibody. Specific binding was detectedusing the Nova Red substrate kit.

Results

Generation of CMAH null mice—To investigate physiological functions ofNeu5Gc in vivo, mice were generated that were deficient inCMP-N-acetylneuraminic acid hydroxylase (CMAH) activity. The originalgoal was to produce CMAH conditional mutant mice using the Cre/loxPsystem. Thus, a targeting vector, pFlox-Ex-SL, was first prepared inwhich exon 6 of CMAH and the TK/Neo cassette, are flanked by loxP sites(FIG. 14). The targeting vector was electroporated into ES cells and 1,out of 245 clones was identified as a homologous recombinant by Southernblot and PCR analysis. This clone was transfected with a Cre-recombinaseexpression vector and 0 out of 150 clones had undergone type IIrecombination that would only delete the TK/Neo cassette. Due tosubsequent difficulties obtaining type II recombinants, we chose toselect only two type I recombinants (where both the exon 6 and theTK/Neo cassette were deleted) for blastocyst injection into C57BL/6females. Chimeric mice were bred and one clone achieved germlinetransmission.

Mice deficient in CMAH were viable and fertile and showed no grossmorphological or histological abnormalities in many organs studied, andtheir growth was equivalent to that of wild-type littermates (data notshown). Transmission of the null allele occurred at the expectedMendelian frequency in pups resulting from heterozygous breeding asdepicted below in Table 1:

TABLE 1 Genotypes of litters from intercrosses of CMAH heterozygous miceNo. (%) of pups with genotype Wild type (+/+) 12 (20) Heterozygous (+/−)38 (63) Homozygous (−/−) 10 (17)

CMAH null mice are deficient in Neu5Gc expression. To verify that CMAHnull mice were deficient in CMAH expression, the mice were analyzed forNeu5Gc expression by immunohistochemistry using a chicken antibodyspecific for Neu5Gc, and biochemically, by derivatization with1,2-diamino-4,5-methylene dioxybenzene (DMB) and HPLC analysis. Usingthe anti-Neu5Gc antibody, there was no staining in any tissues exceptfor the mucinous secretions of the small intestine and colon. To confirmthe lack of Neu5Gc in these tissues as well as those not stainedpositive, various tissues were homogenized and the sialic acids inglycopeptides and lipid extracts were released by acid and purified byion exchange chromatography, derivatized with DMB and analyzed by HPLC(DMB-HPLC). No Neu5Gc was detectable in tissues staining negative withthe anti-Neu5Gc antibody (data not shown), nor could we find evidencefor presence of Neu5Gc in the intestine or the colon (FIG. 15). Thus, itis concluded that the staining of the mucin-rich regions of theintestine was non-specific. Furthermore, no Neu5Gc could be detected inthe plasma or the milk of the CMAH null mouse (FIG. 15.)

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

1. A viable and fertile transgenic mouse that a) comprises a genomehaving a homozygous mutation of the cytidine-5′-monophosphate N-acetylneuraminic acid hydrolase (CMAH) gene, b) lacks expression ofenzymatically active CMAH protein encoded by said CMAH gene, and c)lacks N-glycolylneuraminic acid (Neu5Gc) in one or more body fluid ortissue.
 2. The mouse of claim 1, wherein said body fluid is selectedfrom the group consisting of serum and milk.
 3. The mouse of claim 1,wherein said tissue comprises muscle.